JP2019088324A - Modified T lymphocytes - Google Patents

Abstract

To provide modified T lymphocytes for treating diseases, such as cancer.SOLUTION: Provided is a T lymphocyte comprising an artificial cell death polypeptide comprising an apoptosis-inducing domain, the artificial cell death polypeptide being a transmembrane protein comprising an extracellular domain comprising a CD20 epitope, a transmembrane domain, and an intracellular domain comprising the apoptosis induction domain, the apoptosis induction domain being caspase 3, caspase 8 or caspase 9, the polypeptide being capable dimerization using an anti-CD20 antibody which binds to the CD20 epitope, and an apoptosis induction signal being generated in the T lymphocytes when the antibody dimerizes the polypeptide.SELECTED DRAWING: None

Description

This application claims priority to US Provisional Patent Application No. 61 / 794,294, filed March 15, 2013, the disclosure of which is incorporated herein by reference in its entirety.

(1. Field)
The disclosure herein relates to the field of immunology, and more specifically to the modification of T lymphocytes or other immune cells.

(2. Background)
T lymphocytes recognize and interact with specific antigens, including tumor associated antigens or tumor specific antigens. Because T lymphocytes can kill tumor cells, antigen-specific T lymphocytes, or one or more chimeric antigen receptors (CAR; eg, US Pat. No. 7,741, Eshhar) for the past 25 years.
, 465; see US Patent Application Publication No. 2012/0093842 (see Eshhar) (US Patent Application Publication No. 2012/0093842). There has been great interest in targeting tumor cells by T lymphocytes either of which have been genetically modified to express . However, considering that T lymphocytes can kill not only tumor cells presenting a specific antigen but also normal cells presenting the same antigen, the off-target effect should be harmful to the patient in the unlikely event If this proves to be, it is desirable to incorporate in T lymphocytes a safety mechanism that allows for the rapid killing of the cells after administration to a patient.

A system for killing T cells has been described (Straathof et al. (2005) Blood 105).
(11): 4247-4254) As this system was dependent on specific and difficult protein modifications, this system has become undesirable for practical applications. Thus, there is a need in the art for a relatively simple and easy-to-build safety system for rapidly killing therapeutic T lymphocytes. Provided herein are T lymphocytes comprising such a safety system.

(3. Overview)
Provided herein are multimerizing agents, such as multimerizing with dimerizing agents,
For example, when dimerized, an apoptosis induction signal is generated in cells expressing the polypeptide, eg, T lymphocytes, resulting in, for example, cell death by apoptosis.
Genetically modified cells, eg, immune cells, eg, T lymphocytes, comprising multimerizable, eg, dimerizable artificial polypeptides (herein referred to as "cell death polypeptides") For example, human T lymphocytes. While not wishing to be bound by any particular mechanism or theory, a sufficient number of the plurality of cell death polypeptides of the cell are produced thereby when multimerized, eg, dimerized, The collective apoptosis induction signal is believed to be sufficient to kill the cells, eg, to cause the cells to undergo apoptosis.

The cell death polypeptide provided herein can be used with any cell, in particular any mammalian cell, such as any human cell. For example, such cell death polypeptides provide, for example, useful safety properties for cell therapeutics. Thus, the cell death polypeptide may be important, for example, for drug products that include cellular therapeutics, such as chimeric antigen receptor expressing CAR T lymphocytes. Because, if the cell death polypeptide is desired to rapidly kill cell therapeutics, eg, T lymphocytes,
For example, if administration of the cells results in any undesired or deleterious effects in the patient receiving it, or if the presence of a cellular therapeutic, such as T lymphocytes, in the subject is no longer needed, such rapid It is because that makes it possible to Thus, in one embodiment, the cell death polypeptide provided herein comprises any administrable cell, such as a cellular therapeutic, eg, a mammalian cellular therapeutic, eg, a human cellular therapeutic Can be used with Non-limiting examples of cells in which the cell death polypeptide and multimerization or dimerization factor may be used include natural killer (NK) cells, dendritic cells (DC), placental stem cells (eg, the disclosure thereof) No. 7,468,2 which is incorporated herein by reference in its entirety.
76; placental stem cells disclosed in 8,057,788 and 8,202,703), mesenchymal stem cells derived from cord blood, placental blood, peripheral blood, bone marrow, dental pulp, adipose tissue, osteochondral tissue, etc .; Examples include, but are not limited to, stem cells, embryonic germ cells, neural crest stem cells, neural stem cells, and differentiated cells such as fibroblasts. The cell death polypeptide and multimerization or dimerization factor can also be used in tumor cell lines, for example for the purpose of animal model experiments.

Typically, the cell death polypeptide uses an administrable multimerization or dimerization factor, such as a small molecule, a polypeptide (other than a cell death polypeptide), such as an antibody, an oligonucleotide, or a polysaccharide. Can be multimerized or dimerized. The cell death polypeptide is FK506
It does not contain a binding protein (FKBP), a functional fragment thereof, or a modified form thereof, and the multimerizing agent or dimerizing agent is not an FKBP ligand.

In a first aspect, provided herein is a cell comprising a cell death polypeptide comprising an apoptosis inducing domain, such as a T lymphocyte, wherein said cell death polypeptide is multimerized. An agent can be multimerized, and when the multimerizing agent dimerizes the polypeptide, an apoptosis induction signal is generated in the cell. In a specific embodiment, the multimerizing agent is a dimerizing agent; ie, the multimerizing agent dimerizes the cell death polypeptide. In another specific embodiment, an apoptosis induction signal is generated in the cell when the dimerization factor dimerizes the polypeptide.

In one embodiment, the cell death polypeptide is a transmembrane polypeptide comprising an extracellular domain, a transmembrane domain, and an intracellular domain comprising the apoptosis induction domain. In certain embodiments, the apoptosis-inducing domain of the cell death polypeptide is a caspase, eg, caspase 9, caspase 8 or caspase 3, eg, human caspase 9, caspase 8 or caspase 3 or Including.

In one embodiment, the dimerization factor is a cell death polypeptide, eg, a polypeptide comprising at least two sites that specifically bind to the extracellular domain of the cell death polypeptide. In certain embodiments, the polypeptide is an antibody, eg, an antibody that comprises at least two epitopes or mimotope binding sites. In one embodiment, only the antigen binding domain of the antibody is used as a multimerization or dimerization agent. In one embodiment, the extracellular domain of the cell death polypeptide comprises at least one epitope or mimotope to which the antibody specifically binds. In a specific embodiment, the antibody is a bispecific antibody comprising two different epitopes or mimotopes binding sites that bind to two different epitopes or mimotopes present on the extracellular domain of a cell death polypeptide. In one embodiment, the antibody is an IgG or an IgM antibody. In certain embodiments, antibodies useful as multimerizing or dimerizing agents are those that have been approved by the U.S. Food and Drug Administration for any use.

In one embodiment, an antibody useful as a multimerizing or dimerizing agent is a CD20 epitope or mimotope, eg, an antibody that specifically binds to a human CD20 epitope or mimotope, and the extracellular domain of a cell death polypeptide Contains a CD20 epitope or mimotope to which the antibody specifically binds. In a specific embodiment, the antibody is rituximab and the extracellular domain of the cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that specifically binds to the rituximab. In another specific embodiment, the antibody is tosituzumab and the extracellular domain of the cell death polypeptide specifically binds to the tosituzumab
It contains the CD20 epitope or the CD20 mimotope. In another embodiment, the antibody is ibritumomab and the extracellular domain of the cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that specifically binds to the ibritumomab. In yet another embodiment, the antibody is ofatumumab and the extracellular domain of the cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that specifically binds to the ofatumumab.

In another specific embodiment, the antibody is alemtuzumab and the extracellular domain of the cell death polypeptide comprises a CD52 epitope or a CD52 mimotope that specifically binds to the alemtuzumab. In another embodiment, the antibody is basiliximab and the extracellular domain of the cell death polypeptide is a CD25 epitope that specifically binds to the basiliximab or
Includes CD25 mimotope. In another embodiment, the antibody is daclizumab and the extracellular domain of the cell death polypeptide is a CD25 epitope specifically binding to daclizumab or
Includes CD25 mimotope. In yet another embodiment, the antibody is brentuximab and the extracellular domain of a cell death polypeptide specifically binds CD30 to said brentuximab
It contains an epitope or CD30 mimotope. In another embodiment, the antibody is belimumab and the extracellular domain of the cell death polypeptide comprises a B cell activating factor (BAFF) epitope or a BAFF mimotope that specifically binds to the belimumab. In another embodiment, the antibody is cetuximab and the extracellular domain of the cell death polypeptide comprises an epidermal growth factor receptor (EGFR) epitope or an EGFR mimotope that specifically binds to the cetuximab. In another embodiment, the antibody is panitumumab and the extracellular domain of the cell death polypeptide comprises an epidermal growth factor receptor (EGFR) epitope or EGFR mimotope that specifically binds to the panitumumab. In another embodiment, the antibody is efalizumab and the extracellular domain of the cell death polypeptide is an epitope of CD11a or C that specifically binds to the efalizumab
Includes the D11a mimotope. In yet another embodiment, the antibody is ipilimumab,
The extracellular domain of the cell death polypeptide comprises a CD152 epitope or a CD152 mimotope that specifically binds to the ipilimumab. In yet another embodiment, the antibody is natalizumab and the extracellular domain of the cell death polypeptide comprises an epitope of alpha 4 integrin or a mimotope of alpha 4 integrin that specifically binds to said natalizumab. In another embodiment, the antibody is basiliximab and the extracellular domain of the cell death polypeptide comprises a CD25 epitope or a CD25 mimotope that specifically binds to the basiliximab.

In one embodiment, when the multimerizing agent or the dimerizing agent is bound to at least two cell death polypeptides, dimerization or multimerization of the cell death polypeptide occurs, eg, the cell death Dimerization or multimerization of the polypeptide occurs. In certain embodiments, the extracellular domain of a cell death polypeptide is a receptor or a ligand binding portion thereof, or
Or these are included. In a specific embodiment, the multimerizing agent or dimerizing agent is or comprises at least two ligands of said receptor or its ligand binding portion. In another specific embodiment, the multimerizing agent or dimerizing agent binds to the receptor or the ligand binding portion thereof on two of the cell death polypeptides, and the polypeptide is a multimer. For example, the intracellular domain of the polypeptide is multimerized or dimerized. In certain embodiments, the cell death polypeptide comprises an intracellular domain comprising a caspase domain, wherein multimerization or dimerization of the caspase domain occurs.
In a specific embodiment, said multimerisation or dimerisation, eg multimerisation or dimerisation of intracellular domains, eg multimerisation or dimerisation of caspase domains, is an apoptosis induction signal Is elicited by the cells, eg, T lymphocytes.

In a specific embodiment, dimerization of the cell death polypeptide occurs when the antibody specifically binds to an epitope or mimotope of at least two cell death polypeptides,
For example, dimerization of the intracellular domain of the cell death polypeptide occurs. In certain embodiments, the cell death polypeptide comprises an intracellular domain comprising a caspase domain, wherein dimerization of the caspase domain occurs. In a specific embodiment, the dimerization, eg, intracellular domain dimerization, eg, caspase domain dimerization, elicits an apoptosis induction signal in the cells, eg, T lymphocytes. Do.

In certain other embodiments of cells, eg, T lymphocytes, the extracellular domain of the cell death polypeptide comprises a ligand for the receptor. In a specific embodiment, the multimerizing agent or dimerizing agent comprises at least two receptors or ligand binding portions thereof that bind to the ligand. In a specific embodiment, when the multimerizing agent or dimerizing agent is bound to the receptor or the ligand binding portion thereof on at least two of the cell death polypeptides, the polypeptide is multimeric. Or dimerize. In a specific embodiment, an apoptosis induction signal is generated in the cell when the cell death polypeptide is multimerized or dimerized.

In certain other embodiments, the extracellular domain of a cell death polypeptide comprises an artificial oligonucleotide sequence. For example, in certain embodiments, a modified cell, eg, a T lymphocyte, comprises a cell death polypeptide comprising an extracellular domain comprising an artificial oligonucleotide sequence. In a specific embodiment, the multimerizing or dimerizing agent is at least one multimerizing or dimerizing optionally comprising a first oligonucleotide and a second oligonucleotide, optionally connected by a linker. An oligonucleotide or comprising said multimerized or dimerized oligonucleotide, wherein said first oligonucleotide and said second oligonucleotide are complementary to said artificial oligonucleotide sequence. In one specific embodiment, the first oligonucleotide and the second oligonucleotide have the same sequence. In a specific embodiment, the first oligonucleotide and the second oligonucleotide are connected in the form of a head and a head or a tail and a tail. In a specific embodiment, when said multimerizing or dimerizing oligonucleotide of said multimerizing agent or dimerizing agent is hybridized to an artificial oligonucleotide sequence of two of said cell death polypeptides: The cell death polypeptide is multimerized or dimerized. In another specific embodiment, an apoptosis induction signal is generated in the cell when the cell death polypeptide is multimerized or dimerized. In a specific embodiment, the cell death polypeptide comprises an intracellular caspase domain, wherein the intracellular caspase domain is multimerized or dimerized:
An apoptosis induction signal is generated in the cell.

In certain other embodiments of cells, eg, T lymphocytes, the multimerization or dimerization agent is an artificial polypeptide comprising two or more binding domains connected by one or more linkers.

In a specific embodiment, provided herein is an extracellular domain comprising an epitope, a transmembrane domain and an intracellular domain comprising caspase 9, eg human caspase 9, or a functional fragment thereof. A cell, such as a T lymphocyte, comprising a cell death polypeptide comprising In another specific embodiment, provided herein is a cell comprising an extracellular domain comprising a receptor or ligand binding portion thereof and caspase 9, eg human caspase 9, or a functional fragment thereof A cell comprising an artificial polypeptide comprising an internal domain, eg,
It is a T lymphocyte. In another specific embodiment, provided herein is an extracellular domain comprising a ligand or receptor binding portion thereof, wherein the ligand binds to the receptor or ligand binding portion thereof. A cell, eg, a T lymphocyte, comprising an artificial polypeptide comprising: and caspase 9, eg, an intracellular domain comprising human caspase 9, or a functional fragment thereof. In a specific embodiment, the cell is a T lymphocyte.

In another aspect, provided herein is a cell death polypeptide comprising (a) an extracellular domain comprising an epitope, a transmembrane domain, and a intracellular domain comprising a caspase or a functional part thereof. And (b) cells comprising a dimerization agent comprising two epitope binding or mimotope binding domains that cause the polypeptide to dimerize when contacted with the two cell death polypeptides, wherein: The caspase is caspase 3, caspase 8 or caspase 9, such as human caspase 3, caspase 8 or caspase 9, and the dimerization causes an apoptosis induction signal to be generated in the cell, eg a cell , T lymphocyte safety system. In a specific embodiment, the cell is a T lymphocyte.

In another embodiment, provided herein is an artificial polypeptide comprising (a) an extracellular domain comprising a receptor or ligand binding portion thereof and a intracellular domain comprising a caspase or functional portion thereof. A cell comprising; and (b) 2 bound to the receptor or its ligand binding portion
The dimerization agent comprises a dimerization agent comprising two ligands, wherein when the dimerization agent is contacted with two of the polypeptides, the dimerization agent dimerizes the polypeptide, The caspase may be caspase 3, caspase 8 or caspase 9, for example human caspase 3, caspase
8 or Caspase 9, and the dimerization is a safety system of cells, such as T lymphocytes, which generate an apoptosis induction signal in the cells. In a specific embodiment, the cell is a T lymphocyte.

In another embodiment, provided herein is an artificial cell death polypeptide comprising (a) an extracellular domain comprising a ligand or a receptor binding portion thereof and a intracellular domain comprising a caspase or a functional portion thereof And (b) a dimerization agent comprising two receptors which bind to the ligand or its receptor binding portion, or a ligand binding portion thereof, wherein the dimerization agent is When contacted with one of the polypeptides, the dimerization factor dimerizes the polypeptide, and the caspases are caspase 3, caspase 8, or caspase 9, such as human caspase 3, caspase 8, Or Caspase 9, and the dimerization is a safety system of cells, such as T lymphocytes, which generate an apoptosis induction signal in the cells. In a specific embodiment, the cell is a T lymphocyte.

In a specific embodiment of any of the embodiments herein, when a plurality of said apoptosis induction signals are generated in said cells, eg T lymphocytes, said signals kill the cells. It is enough. In a specific embodiment, the cell is a T lymphocyte.

In another aspect, further provided herein is a method of killing a cell, eg, a T lymphocyte, wherein the cell comprises a plurality of artificial cell death polypeptides each comprising an apoptosis induction domain. The cell death polypeptide is multimerizable or dimerizable using a multimerizing agent or dimerizing agent that is not an FK506 binding protein (FKBP) ligand, and the multimerizing agent or dimerizing agent When the dimerization agent multimerizes or dimerizes the polypeptide,
Apoptosis inducing signal is generated in said T lymphocytes, and the cells are collectively sufficient to multimerize or dimerize said plurality of said cell death polypeptides to kill said cells. Contacting with an amount of said multimerizing agent or dimerizing agent sufficient to generate an apoptosis induction signal. In one embodiment, the cell death polypeptide is a transmembrane polypeptide comprising an extracellular domain, a transmembrane domain, and an intracellular domain comprising the apoptosis induction domain. In a specific embodiment, the apoptosis-inducing domain of the polypeptide is a caspase, such as caspase 3, caspase 8,
Or caspase 9, for example human caspase 9, caspase 8 or caspase 3 or include them. In a specific embodiment, the multimerizing agent or dimerizing agent is a protein, an oligonucleotide, or a polysaccharide. In a specific embodiment,
The cells are T lymphocytes.

In one embodiment, the multimerizing agent or dimerizing agent is a protein, an oligonucleotide, or a polysaccharide. In a specific embodiment, the multimerizing agent or dimerizing agent is a cell death polypeptide, eg, a polypeptide comprising at least two sites that specifically bind to the extracellular domain of the cell death polypeptide. is there. In certain embodiments, the polypeptide is an antibody, such as an antibody comprising at least two epitope binding sites or at least two mimotope binding sites. In one embodiment, the extracellular domain of the cell death polypeptide comprises at least one epitope or mimotope to which the antibody specifically binds. In a specific embodiment, the antibody is a bispecific antibody comprising two different epitopes or mimotopes binding sites that bind to two different epitopes or mimotopes present on the extracellular domain of a cell death polypeptide. In one embodiment, the antibody is an IgG or an IgM antibody. In a specific embodiment, antibodies useful as multimerizing or dimerizing agents are those that have been approved by the U.S. Food and Drug Administration for any use.

In a specific embodiment, when said multimerizing agent or dimerizing agent is an antibody, said antibody is an antibody that specifically binds to a CD20 epitope or mimotope, such as a human CD20 epitope or mimotope, The extracellular domain of the cell death polypeptide comprises a CD20 epitope or mimotope to which the antibody specifically binds. In a specific embodiment, when said multimerizing agent or dimerizing agent is an antibody, said antibody is rituximab, and said extracellular domain of a cell death polypeptide binds CD20 to said rituximab. An epitope or CD20 mimotope; the antibody is tosituzumab, and the extracellular domain of the cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that binds to the tosituzumab; the antibody is ibritumomab and a cell Whether the extracellular domain of the death polypeptide comprises a CD20 epitope or a CD20 mimotope that binds to the ibritumomab; the antibody is ofatumumab and the extracellular domain of the cell death polypeptide is a CD20 epitope binding to the ofatumumab Or contains the CD20 mimotope; the antibody is alemtuzumab and And the extracellular domain of cell death polypeptide binds to said alemtuzumab
Does the antibody contain basiliximab and the extracellular domain of the cell death polypeptide contains the CD25 epitope or the CD25 mimotope that binds to the basiliximab; the antibody is daclizumab, and The extracellular domain of a cell death polypeptide comprises a CD25 epitope or CD25 mimotope that binds to daclizumab; the antibody is brentuximab and the extracellular domain of a cell death polypeptide is the brentuximab Contains a CD30 epitope or a CD30 mimotope that binds to said; said antibody is belimumab and said extracellular domain of a cell death polypeptide comprises a B cell activating factor (BAFF) epitope or a BAFF mimotope binding to said belimumab The antibody is cetuximab and a cell death polypeptide The of the extracellular domain comprises or epidermal growth factor receptor (EGFR) epitope or EGFR mimotope binds to the cetuximab;
Is the antibody panitumumab and does the extracellular domain of the cell death polypeptide contain an epidermal growth factor receptor (EGFR) epitope or EGFR mimotope that binds to the panitumumab?
The antibody is efalizumab and the extracellular domain of the cell death polypeptide comprises an epitope of CD11a or a mimotope of CD11a that binds to the efalizumab; the antibody is ipilimumab and the cell death polypeptide Does the extracellular domain contain the CD152 epitope or CD152 mimotope that binds to the ipilimumab; the antibody is natalizumab and the extracellular domain of the cell death polypeptide is the epitope of the α4 integrin or the α4 integrin that binds to the natalizumab Or the antibody is basiliximab, and the extracellular domain of the cell death polypeptide comprises a CD25 epitope or a CD25 mimotope that binds to the basiliximab. In a specific embodiment of any of the above embodiments, the intracellular domain of the polypeptide, and / or the intracellular domain, when the antibody is bound to the epitope or mimotope on at least two of the cell death polypeptide Each caspase in the intracellular domain is multimerized or dimerized. In a specific embodiment, when the antibody specifically binds to an epitope or mimotope of at least two cell death polypeptides, dimerization of the cell death polypeptide occurs, eg, the cell death polypeptide Dimerization of the intracellular domain of In certain embodiments, the cell death polypeptide comprises an intracellular domain comprising a caspase domain, wherein dimerization of the caspase domain occurs. In a specific embodiment, the dimerization, eg, intracellular domain dimerization, eg, caspase domain dimerization, elicits an apoptosis induction signal in the cells, eg, T lymphocytes. Do.

In a specific embodiment, the extracellular domain of the cell death polypeptide is or comprises a receptor or a ligand binding portion thereof. In such embodiments, the multimerizing agent or dimerizing agent comprises at least two ligands of the receptor or its ligand binding portion. In a specific embodiment, when the multimerizing agent or dimerizing agent is bound to the receptor or the ligand binding portion thereof on at least two of the cell death polypeptides, the polypeptide is multimeric. Or dimerize. In a specific embodiment, the multimerization or dimerization of the polypeptide elicits an apoptosis induction signal in the cell, eg, a T lymphocyte.

In a specific embodiment, the extracellular domain of the cell death polypeptide comprises a ligand for a receptor. In such embodiments, the multimerizing agent or dimerizing agent is
And at least two receptors that bind to said ligand or their ligand binding moieties. In a specific embodiment, when the multimerizing agent or dimerizing agent is bound to the receptor or the ligand binding portion thereof on two or more of the polypeptides, the intracellular domain of the polypeptide and And / or the caspase domain of the polypeptide is multimerized or dimerized. In a specific embodiment, the multimerization or dimerization of the intracellular domain and / or caspase domain elicits an apoptosis inducing signal generated in the cell, eg, T lymphocytes.

In one embodiment, the extracellular domain of the cell death polypeptide comprises an artificial oligonucleotide sequence. For example, in certain embodiments, the cell death polypeptide comprises an extracellular domain comprising an artificial oligonucleotide sequence. In a specific embodiment, the multimerizing or dimerizing agent is at least one multimerizing or dimerizing optionally comprising a first oligonucleotide and a second oligonucleotide, optionally connected by a linker. An oligonucleotide or comprising said multimerized or dimerized oligonucleotide, wherein said first oligonucleotide and said second oligonucleotide are complementary to said artificial oligonucleotide sequence. In one specific embodiment, the first oligonucleotide and the second oligonucleotide have the same sequence. In a specific embodiment, the first oligonucleotide and the second oligonucleotide are connected in the form of a head and a head or a tail and a tail. In a specific embodiment, when said multimerizing or dimerizing oligonucleotide of said multimerizing agent or dimerizing agent is hybridized to an artificial oligonucleotide sequence of two of said cell death polypeptides: The cell death polypeptide is multimerized or dimerized. In another specific embodiment, an apoptosis induction signal is generated in the cell when the cell death polypeptide is multimerized or dimerized. In a specific embodiment, the cell death polypeptide comprises an intracellular caspase domain, and an apoptosis inducing signal is generated in the cell when the intracellular caspase domain is multimerized or dimerized. In a specific embodiment, the cell is a T lymphocyte.

In a specific embodiment of any of the embodiments herein, when the plurality of apoptosis induction signals are generated in the T lymphocytes, the signals kill the cells, eg, T lymphocytes. Is enough.

In another embodiment, the multimerizing agent or dimerizing agent is an artificial polypeptide comprising two or more binding domains connected by one or more linkers.

In a specific embodiment, provided herein is a method of killing a cell, eg, a T lymphocyte, wherein the cell comprises a plurality of artificial cell deaths each comprising a caspase or a functional part thereof. A polypeptide, wherein the caspase is caspase 3, caspase 8, or caspase 9 (eg, human caspase 3, caspase 8 or caspase 9), and the cell death polypeptide can be dimerized using an antibody And, when the antibody dimerizes the polypeptide, an apoptosis induction signal is generated in the cell, and the plurality of the plurality is sufficient to dimerize the cell. And D. contacting the artificial polypeptide with an amount of the antibody sufficient to generate a collective apoptosis induction signal sufficient to kill the cell. In a specific embodiment, the cell is a T lymphocyte.

In another specific embodiment, provided herein is a method of killing a cell, eg, a T lymphocyte, wherein the cell comprises a plurality of artificial cells each comprising a caspase or a functional portion thereof. A cell death polypeptide, wherein the caspase is caspase 3, caspase 8 or caspase 9 (e.g. human caspase 3, caspase 8 or caspase 9) and the plurality of artificial polypeptides are each bound to a ligand And / or an extracellular domain comprising a ligand binding portion thereof, wherein the polypeptide is dimerizable with a dimerization agent comprising two of the ligands, and the dimerization agent When the antibody dimerizes two of the polypeptides, an apoptosis-inducing signal is generated in the cells, and a sufficient number of the plurality of artificial cell deaths are sufficient to dimerize the cells. Ripepuchido is dimerized, comprising contacting a sufficient amount of antibody to generate a sufficient collective apoptosis-inducing signal to kill the cells, it is a method. In a specific embodiment, the cell is a T lymphocyte.

In another specific embodiment, provided herein is a method of killing a cell, eg, a T lymphocyte, wherein the cell comprises a plurality of artificial cells each comprising a caspase or a functional portion thereof. A cell death polypeptide, wherein the caspase is caspase 3, caspase 8 or caspase 9 (e.g. human caspase 3, caspase 8 or caspase 9) and the plurality of artificial polypeptides are each a receptor or Comprising an extracellular domain comprising a ligand which binds to its ligand binding portion or its receptor binding portion, said polypeptide being dimerized using a dimerization agent comprising two of said receptors or its ligand binding portion Are characterized in that when the dimerization factor dimerizes two of the polypeptides, an apoptosis induction signal is generated in the cells, The amount of the dimers is sufficient to dimerize a sufficient number of the plurality of artificial polypeptides to dimerize and to generate a collective apoptosis induction signal sufficient to kill the cells. A method comprising contacting with a somatizing agent. In a specific embodiment, the cell is a T lymphocyte.

In another specific embodiment, provided herein is a method of killing a cell, eg, a T lymphocyte, wherein the cell comprises a plurality of artificial cells each comprising a caspase or a functional portion thereof. A cell death polypeptide, wherein said caspase is caspase 3, caspase 8 or caspase 9 (e.g. human caspase 3, caspase 8 or caspase 9), said plurality of artificial polypeptides each being an artificial oligonucleotide A plurality of polypeptides comprising an extracellular domain comprising: and a plurality of said polypeptides being dimerizable with a dimerization factor comprising an oligonucleotide comprising a first oligonucleotide and a second oligonucleotide; One oligonucleotide and the second oligonucleotide have the same nucleotide sequence, the first oligonucleotide and the second oligonucleotide Reotide is optionally connected by a linker, the first oligonucleotide and the second oligonucleotide are complementary to the artificial oligonucleotide in the extracellular domain of the polypeptide, and the dimer When the growth factor dimerizes two of the cell death polypeptides, an apoptosis-inducing signal is generated in the cell, and a sufficient number of the plurality is sufficient to dimerize the cell. A method comprising dimerizing an artificial polypeptide of SEQ ID NO: 1 and contacting with an amount of said dimerization factor sufficient to generate a collective apoptosis induction signal sufficient to kill said cells. . In a specific embodiment, the cell is a T lymphocyte.

In certain embodiments, cells that are killed according to the methods described herein (eg, T
Lymphocytes) comprise a polypeptide that acts to target the cell to a particular antigen, such as a tumor associated antigen or a tumor specific antigen, wherein the polypeptide when bound to the antigen Kill cells presenting the antigen, eg, a chimeric antigen receptor (CAR). T lymphocytes, including CAR, are referred to herein as CAR-T lymphocytes. The chimeric antigen receptor is usually (i) an intracellular domain (eg, a cytoplasmic domain) of an endogenous protein which is expressed on the surface of the lymphocyte and induces activation and / or proliferation of the lymphocyte (ii) membrane The penetration domain, as well as (iii) the antigen of interest, eg, an extracellular domain that binds to a tumor associated antigen or tumor specific antigen. CAR-T lymphocytes usually also contain one or more costimulatory domains. In certain embodiments, CAR-T lymphocytes comprise at least two CAR polypeptides, at least one of which provides a primary stimulation signal to CAR-T lymphocytes, at least one of which is CAR- Provides costimulatory signals to T lymphocytes. Contains a cell death polypeptide and C
CAR-T lymphocytes are provided below, including certain embodiments of the AR.

In another aspect, provided herein is a method of treating an individual having a disease or disorder, wherein the disease or disorder is characterized or characterized by cells expressing an antigen. A method comprising: administering to said individual a cell expressing a polypeptide as described herein, eg, a T lymphocyte. In certain embodiments, the modified cells described herein, such as modified T lymphocytes, when administered to a subject in need thereof, the multimerization factor and the cell death polypeptide selected Combinations are selected such that they are compatible with the patient population (or subpopulation) to which the cells, eg, T lymphocytes, have been administered. By way of example only, where the multimerization factor selected is the antibody rituximab, in certain embodiments, the patient population is an individual with B-cell cancer, eg, B-cell lymphoma.

(4. Detailed description)
(4.1. Cells containing cell death polypeptide)
Provided herein are multimerizing agents, such as multimerizing with dimerizing agents,
For example, when dimerized, an apoptosis induction signal is generated in cells expressing the polypeptide, eg, T lymphocytes, resulting in, for example, cell death by apoptosis.
Genetically modified cells, eg, immune cells, eg, T lymphocytes, comprising multimerizable, eg, dimerizable artificial polypeptides (herein referred to as "cell death polypeptides") For example, human T lymphocytes. While not wishing to be bound by any particular mechanism or theory, a sufficient number of the plurality of cell death polypeptides of the cell are produced thereby when multimerized, eg, dimerized, The collective apoptosis induction signal is believed to be sufficient to kill the cells, eg, to cause the cells to undergo apoptosis. In a specific embodiment, the genetically modified cells provided herein are T lymphocytes.

In certain embodiments, the cell death polypeptide is an administrable multimerizing agent or dimerizing agent, such as a protein (eg, an antibody, a receptor or a ligand binding portion thereof, a ligand or a receptor binding portion thereof) And can be multimerized or dimerized by oligonucleotides or the like. In one embodiment, the multimerizing agent is not a small molecule. The multimerization or dimerization factor can be used to kill T lymphocytes containing the cell death polypeptide either in vitro or in vivo.

Thus, in a first aspect, provided herein is a T lymphocyte comprising an artificial polypeptide (cell death polypeptide) comprising an apoptosis inducing domain, wherein said cell death polypeptide is Multimerization is possible using multimerization factor, which is FK
An apoptosis-inducing signal is generated in the T lymphocytes when not the 506 binding protein (FKBP) ligand and the multimerization factor causes the polypeptide to multimerize. In a specific embodiment, the multimerizing agent is a dimerizing agent; ie, the multimerizing agent is
The cell death polypeptide is dimerized. In another specific embodiment, an apoptosis induction signal is generated in the T lymphocytes when the dimerization factor dimerizes the cell death polypeptide. The cell death polypeptide does not comprise FK506 binding protein, a functional fragment thereof, or a modified form thereof.

In one embodiment, the cell death polypeptide is a transmembrane polypeptide comprising an extracellular domain, a transmembrane domain, and an intracellular domain comprising the apoptosis induction domain. In certain embodiments, the apoptosis induction domain of the cell death polypeptide can be, for example, any protein or portion thereof that elicits an apoptosis induction signal in the cell when dimerized. In certain embodiments, the apoptosis induction domain is any caspase that homodimerizes, preferably a caspase, such as caspase 9, caspase 8, or caspase 3 (eg, human caspase 9, caspase 8,
Or caspase 3) or include them. Human caspase 9, human caspase 8
And amino acid sequences of human caspases, including human caspase 3, are well known in the art. For example, human caspase 3 is assigned to NCBI gene ID: 836; human caspase 8 is assigned to NCBI gene ID: 841; human caspase 9 is NCBI gene ID:
It is assigned to 842. In certain embodiments, the intracellular domain that is a caspase domain or an extracellular domain that includes an epitope or mimotope that is a caspase domain is connected by a CD8 alpha stalk or a CD8 beta stalk, at least a portion of which can function as a transmembrane domain It is done.

In one embodiment, the dimerization factor is a cell death polypeptide, eg, a polypeptide comprising at least two sites that specifically bind to the extracellular domain of the cell death polypeptide. In certain embodiments, the polypeptide is an antibody, eg, an antibody that comprises at least two epitopes or mimotope binding sites. In one embodiment, only the antigen binding domain of the antibody is used as a multimerization or dimerization agent. In one embodiment, the extracellular domain of the cell death polypeptide comprises at least one epitope or mimotope to which the antibody specifically binds. In a specific embodiment, the antibody binds to two different epitopes or mimotopes present on the extracellular domain of a cell death polypeptide 2
Bispecific antibodies that contain two different epitopes or mimotope binding sites. In one embodiment, the antibody is an IgG or an IgM antibody. It is also possible to use artificial antibody constructs comprising antibody-derived epitope binding or mimotope binding domains, optionally connected by one or more linkers.

In a specific embodiment, the antibodies useful as multimerizing or dimerizing agents have been approved by government regulatory agencies, such as the US Food and Drug Administration, for any use. This ensures that, for example, the antibody has a known toxicity and patient safety profile when used as a dimerization or multimerization factor. Any combination of antibodies and related targets can be used on the T lymphocytes provided herein. In one embodiment, antibodies useful as multimerizing or dimerizing agents are CD20 epitopes or mimotopes, eg,
An antibody that specifically binds to a human CD20 epitope or mimotope, wherein the extracellular domain of the cell death polypeptide comprises a CD20 epitope or mimotope to which the antibody specifically binds.
In a specific embodiment, the antibody is rituximab and the extracellular domain of the cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that specifically binds to the rituximab. In another specific embodiment, the antibody is tosituzumab and the extracellular domain of the cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that specifically binds to the tosituzumab. In another embodiment, the antibody is ibritumomab and the extracellular domain of the cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that specifically binds to the ibritumomab. In yet another embodiment, the antibody is ofatumumab and the extracellular domain of the cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that specifically binds to the ofatumumab.

In another specific embodiment, the antibody is alemtuzumab and the extracellular domain of the cell death polypeptide comprises a CD52 epitope or a CD52 mimotope that specifically binds to the alemtuzumab. In another embodiment, the antibody is basiliximab and the extracellular domain of the cell death polypeptide is a CD25 epitope that specifically binds to the basiliximab or
Includes CD25 mimotope. In another embodiment, the antibody is daclizumab and the extracellular domain of the cell death polypeptide is a CD25 epitope specifically binding to daclizumab or
Includes CD25 mimotope. In yet another embodiment, the antibody is brentuximab and the extracellular domain of a cell death polypeptide specifically binds CD30 to said brentuximab
It contains an epitope or CD30 mimotope. In another embodiment, the antibody is belimumab and the extracellular domain of the cell death polypeptide comprises a B cell activating factor (BAFF) epitope or a BAFF mimotope that specifically binds to the belimumab. In another embodiment, the antibody is cetuximab and the extracellular domain of the cell death polypeptide comprises an epidermal growth factor receptor (EGFR) epitope or an EGFR mimotope that specifically binds to the cetuximab. In another embodiment, the antibody is panitumumab and the extracellular domain of the cell death polypeptide comprises an epidermal growth factor receptor (EGFR) epitope or EGFR mimotope that specifically binds to the panitumumab. In another embodiment, the antibody is efalizumab and the extracellular domain of the cell death polypeptide is an epitope of CD11a or C that specifically binds to the efalizumab
Includes the D11a mimotope. In yet another embodiment, the antibody is ipilimumab,
The extracellular domain of the cell death polypeptide comprises a CD152 epitope or a CD152 mimotope that specifically binds to the ipilimumab. In yet another embodiment, the antibody is natalizumab and the extracellular domain of the cell death polypeptide comprises an epitope of alpha 4 integrin or a mimotope of alpha 4 integrin that specifically binds to said natalizumab. In another embodiment, the antibody is basiliximab and the extracellular domain of the cell death polypeptide comprises a CD25 epitope or a CD25 mimotope that specifically binds to the basiliximab.

The ligands and receptors can be utilized in the construction of cell death polypeptides provided herein, using multimerizing agents or dimerizing agents comprising the respective ligands of the receptors. The polypeptides can be multimerized or dimerized. In one embodiment, when the multimerizing agent or the dimerizing agent is bound to at least two cell death polypeptides, dimerization or multimerization of the cell death polypeptide occurs, eg, the cell death Dimerization or multimerization of the polypeptide occurs. In certain embodiments, the extracellular domain of a cell death polypeptide is or comprises a receptor or ligand binding portion thereof.
In a specific embodiment, the multimerizing agent or dimerizing agent is or comprises at least two ligands of said receptor or its ligand binding portion. In another specific embodiment, the multimerizing agent or dimerizing agent binds to the receptor or the ligand binding portion thereof on two of the cell death polypeptides, and the polypeptide is a multimer. For example, the intracellular domain of the polypeptide is multimerized or dimerized. In certain embodiments, the cell death polypeptide comprises an intracellular domain comprising a caspase domain, wherein multimerization or dimerization of the caspase domain occurs. In a specific embodiment, said multimerisation or dimerisation, eg multimerisation or dimerisation of intracellular domains, eg multimerisation or dimerisation of caspase domains, is an apoptosis induction signal Is elicited by the cells, eg, T lymphocytes.

In a specific embodiment, dimerization of the cell death polypeptide occurs when the antibody specifically binds to an epitope or mimotope of at least two cell death polypeptides,
For example, dimerization of the intracellular domain of the cell death polypeptide occurs. In certain embodiments, the cell death polypeptide comprises an intracellular domain comprising a caspase domain, wherein dimerization of the caspase domain occurs. In a specific embodiment, the dimerization, eg, intracellular domain dimerization, eg, caspase domain dimerization, elicits an apoptosis induction signal in the cells, eg, T lymphocytes. Do.

In certain other embodiments of cells, eg, T lymphocytes, the extracellular domain of the cell death polypeptide comprises a ligand for the receptor. In a specific embodiment, the multimerizing agent or dimerizing agent comprises at least two receptors or ligand binding portions thereof that bind to the ligand. In a specific embodiment, when the multimerizing agent or dimerizing agent is bound to the receptor or the ligand binding portion thereof on at least two of the cell death polypeptides, the polypeptide is multimeric. Or dimerize. In a specific embodiment, an apoptosis induction signal is generated in the cell when the cell death polypeptide is multimerized or dimerized. In a specific embodiment, the cell is a T lymphocyte.

In certain other embodiments, the extracellular domain of a cell death polypeptide comprises an artificial oligonucleotide sequence. For example, in certain embodiments, a modified cell, eg, a T lymphocyte, comprises a cell death polypeptide comprising an extracellular domain comprising an artificial oligonucleotide sequence. In a specific embodiment, the multimerizing or dimerizing agent is at least one multimerizing or dimerizing optionally comprising a first oligonucleotide and a second oligonucleotide, optionally connected by a linker. An oligonucleotide or comprising said multimerized or dimerized oligonucleotide, wherein said first oligonucleotide and said second oligonucleotide are complementary to said artificial oligonucleotide sequence. In one specific embodiment, the first oligonucleotide and the second oligonucleotide have the same sequence. In a specific embodiment, the first oligonucleotide and the second oligonucleotide are connected in the form of a head and a head or a tail and a tail. In a specific embodiment, when said multimerizing or dimerizing oligonucleotide of said multimerizing agent or dimerizing agent is hybridized to an artificial oligonucleotide sequence of two of said cell death polypeptides: The cell death polypeptide is multimerized or dimerized. In another specific embodiment, an apoptosis induction signal is generated in the cell when the cell death polypeptide is multimerized or dimerized. In a specific embodiment, the cell death polypeptide comprises an intracellular caspase domain, and when the intracellular caspase domain is multimerized or dimerized, an apoptosis induction signal is generated in the cell. In a specific embodiment, the cells are
It is a lymphocyte.

In certain other embodiments of T lymphocytes, the multimerizing or dimerizing agent is an artificial polypeptide comprising two or more binding domains connected by one or more linkers.

In a specific embodiment, provided herein is an extracellular domain comprising an epitope, a transmembrane domain and an intracellular domain comprising caspase 9, eg human caspase 9, or a functional fragment thereof. A cell, such as a T lymphocyte, comprising a cell death polypeptide comprising In another specific embodiment, provided herein is a cell comprising an extracellular domain comprising a receptor or ligand binding portion thereof and caspase 9, eg human caspase 9, or a functional fragment thereof A cell comprising an artificial polypeptide comprising an internal domain, eg,
It is a T lymphocyte. In another specific embodiment, provided herein is an extracellular domain comprising a ligand or receptor binding portion thereof, wherein the ligand binds to the receptor or ligand binding portion thereof. A cell, eg, a T lymphocyte, comprising an artificial polypeptide comprising: and caspase 9, eg, an intracellular domain comprising human caspase 9, or a functional fragment thereof. In a specific embodiment, the cell is a T lymphocyte.

In any of the embodiments herein the modified cell is a T lymphocyte, the T lymphocyte can be a CD4 + T lymphocyte or a CD8 + T lymphocyte. The T lymphocytes can be specific for particular antigens (eg, tumor associated antigens, tumor specific antigens, viral antigens etc) without genetic modification. The T lymphocytes can be genetically modified to express one or more polypeptides that target T lymphocytes to a specific antigen, such as a chimeric antigen receptor.

(4.2. Method for killing cells containing cell death polypeptide)
Cell death polypeptides provided herein can be used in methods of killing cells comprising the cell death polypeptide, such as T lymphocytes. The cell death polypeptide provided herein can be used with any cell, in particular any mammalian cell, such as any human cell. Such cell death polypeptides provide, for example, useful safety properties for cellular therapeutics. Thus, the cell death polypeptide may be important, for example, for drug products that include cellular therapeutics, such as chimeric antigen receptor expressing CAR T lymphocytes. Because, if the cell death polypeptide is desired to rapidly kill cellular therapeutic agents, eg, T lymphocytes, for example, the administration of the cells may cause any undesirable or harmful effects in the patient receiving it. If such effects are to be achieved, or if the presence of a cellular therapeutic in the subject, eg, T lymphocytes, is no longer needed, such rapid killing is possible. Thus, in one embodiment, a cell death polypeptide provided herein is:
It can be used with any administrable cell, for example a cellular therapeutic, such as a mammalian cellular therapeutic, such as a human cellular therapeutic. Non-limiting examples of cells in which the cell death polypeptide and multimerization or dimerization factor may be used include natural killer (NK) cells, dendritic cells (DC), placental stem cells (eg, the disclosure thereof) Placental stem cells disclosed in U.S. Patent Nos. 7,468,276; 8,057,788 and 8,202,703, all of which are incorporated herein by reference), cord blood, placental blood, peripheral blood, bone marrow, dental pulp, fat Mesenchymal stem cells derived from tissues, osteochondral tissues, etc .; embryonic stem cells, embryonic germ cells, neural crest stem cells, neural stem cells, and differentiated cells (
Examples include, but are not limited to, fibroblasts and the like. The cell death polypeptide and multimerization or dimerization factor can also be used in tumor cell lines, for example for the purpose of animal model experiments.

The killing of cells by the cell death polypeptides described herein can be performed in vivo, eg, in the individual to which the cells, eg, T lymphocytes, have been administered, or in vitro, eg, in the laboratory, eg, quality control It can be done as part of the experiment. In one embodiment, provided herein is a method of killing a cell, eg, a T lymphocyte, the cell comprising a plurality of artificial cell death polypeptides each comprising an apoptosis induction domain, The polypeptide is multimerizable or dimerizable using a multimerizing agent or dimerizing agent that is not an FK506 binding protein (FKBP) ligand, and the multimerizing agent or dimerizing When the factor multimerizes or dimerizes the polypeptide, an apoptosis induction signal is generated in the cell, and the plurality of artificial polypeptides are dimerized in the cell, and the cell is characterized. Contacting the multimerizing agent or the dimerizing agent in an amount sufficient to generate a collective apoptosis induction signal sufficient to kill B. In one embodiment, the cell death polypeptide is a transmembrane polypeptide comprising an extracellular domain, a transmembrane domain, and an intracellular domain comprising the apoptosis induction domain. In one embodiment,
An intracellular domain that is a caspase domain or an extracellular domain that includes an epitope or mimotope that is a caspase domain is connected by a CD8α stalk or a CD8β stalk that can function at least in part as a transmembrane domain. In a specific embodiment of the method, is the apoptosis-inducing domain of the polypeptide a caspase, eg caspase 3, caspase 8 or caspase 9 (eg human caspase 9, caspase 8 or caspase 3) Or these are included.

The multimerization factor or dimerization factor used in the method is any compound other than a small molecule capable of dimerizing or multimerizing a cell death polypeptide, such as a protein, an oligonucleotide Or can be a polysaccharide. In one embodiment, the multimerizing agent or dimerizing agent is an antibody, such as an antibody comprising at least two epitope binding sites or at least two mimotope binding sites. In one embodiment, only the antigen binding domain of the antibody is used as a multimerization or dimerization agent. In such embodiments, the extracellular domain of the cell death polypeptide comprises the epitope or mimotope to which the antibody binds. The antibody can be of any titer but preferably it is an IgG
Or IgM antibody.

In a specific embodiment, the antibodies useful as multimerizing or dimerizing agents have been approved by government regulatory agencies, such as the US Food and Drug Administration, for any use. Any combination of antibodies and related targets can be used in the methods of killing T lymphocytes provided herein.

In a specific embodiment, when said multimerizing agent or dimerizing agent is an antibody, said antibody is an antibody that specifically binds to a CD20 epitope or mimotope, such as a human CD20 epitope or mimotope, The extracellular domain of the cell death polypeptide comprises a CD20 epitope or mimotope to which the antibody specifically binds. In a specific embodiment, when said multimerizing agent or dimerizing agent is an antibody, said antibody is rituximab, and said extracellular domain of said cell death polypeptide is CD20 binding to said rituximab. Epitope or
Whether the antibody is tosituzumab and the extracellular domain of the cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that binds to toctumumab; the antibody is ibritumomab; the cell death poly Whether the extracellular domain of the peptide comprises a CD20 epitope or a CD20 mimotope that binds to the ibritumomab; the antibody is ofatumumab and the extracellular domain of the cell death polypeptide is a CD20 epitope or a CD20 that binds to the ofatum Or the antibody is alemtuzumab and the extracellular domain of the cell death polypeptide comprises a CD52 epitope or a CD52 mimotope that binds to the alemtuzumab.

In one specific embodiment, when the multimerizing agent or dimerizing agent is an antibody,
The antibody is basiliximab and the extracellular domain of the cell death polypeptide comprises a CD25 epitope or CD25 mimotope that binds to the basiliximab; the antibody is daclizumab, the extracellular of the cell death polypeptide The domain comprises a CD25 epitope or a CD25 mimotope that binds to said daclizumab; said antibody is brentuximab,
Whether the extracellular domain of the cell death polypeptide comprises a CD30 epitope or a CD30 mimotope that binds to the brentuximab; the antibody is belimumab and the extracellular domain of the cell death polypeptide is the belimumab Does it contain a B cell activating factor (BAFF) epitope or a BAFF mimotope that binds; the antibody is cetuximab and the extracellular domain of the cell death polypeptide is an epidermal growth factor receptor (EGFR) that binds to cetuximab The epitope or EGFR mimotope; the antibody is panitumumab and the extracellular domain of the cell death polypeptide comprises an epidermal growth factor receptor (EGFR) epitope or EGFR mimotope that binds to the panitumumab; the antibody Is efalizumab and the extracellular domain of the cell death polypeptide binds to the efalizumab An epitope of CD11a or a mimotope of CD11a; the antibody is ipilimumab and the extracellular domain of the cell death polypeptide comprises a CD152 epitope or a CD152 mimotope that binds to the ipilimumab
The antibody is natalizumab and the extracellular domain of the cell death polypeptide comprises an epitope of α4 integrin or a mimotope of α4 integrin which binds to the natalizumab; or the antibody is basiliximab and the cell death The extracellular domain of the polypeptide comprises a CD25 epitope or a CD25 mimotope that binds to the basiliximab.

In a specific embodiment of any of the above embodiments, when the antibody is bound to the epitope or mimotope on at least two of the cell death polypeptides, the intracellular domain of the polypeptide and / or the cells Each caspase in the inner domain is multimerized or dimerized. In a specific embodiment, when the antibody specifically binds to an epitope or mimotope of at least two cell death polypeptides, dimerization of the cell death polypeptide occurs, eg, the cell death polypeptide Dimerization of the intracellular domain of In certain embodiments, the cell death polypeptide comprises an intracellular domain comprising a caspase domain, wherein dimerization of the caspase domain occurs. In a specific embodiment, the dimerization, eg, intracellular domain dimerization, eg, caspase domain dimerization, elicits an apoptosis induction signal in the cells, eg, T lymphocytes. Do.

While not intending to be limited by theory, when the antibody is bound to the respective epitope or mimotope on at least two of the polypeptides, the intracellular domain of the polypeptides is multimerized, eg, or dimeric At that time, each caspase in the intracellular domain dimerizes. Dimerization of the polypeptide elicits an apoptosis induction signal in the T lymphocytes.

As described above, the receptor and its respective ligand are used to multimerize or dimerize the cell death polypeptide, thereby achieving killing of cells, eg, T lymphocytes, containing the polypeptide. can do. For example, the extracellular domain of the cell death polypeptide is or comprises a receptor or ligand binding portion thereof. In such embodiments, the multimerizing agent or dimerizing agent is capable of multimerizing or dimerizing the cell death polypeptide, at least two of the receptor or its ligand binding portion. The polypeptide is dimerized when it comprises two ligands and the multimerizing agent or dimerizing agent is bound to the receptor on at least two of the polypeptides or the ligand binding portion thereof . In a preferred embodiment, dimerization of the polypeptide elicits the apoptosis induction signal in the cell.

In another embodiment of the method of killing a cell, eg, a T lymphocyte, the extracellular domain of the cell death polypeptide comprises a ligand for the receptor. In such embodiments of the method, the multimerizing agent or dimerizing agent comprises at least two receptors or ligand binding portions thereof that bind to the ligand. When the multimerizing dimerization factor is bound to the receptor on the two cell death polypeptides or the ligand-binding portion thereof, the intracellular domain of the polypeptide, and thus preferably the caspase domain It is converted to The dimerization of the intracellular domain and the caspase domain preferably elicits an apoptosis-inducing signal generated in the cell.

In certain other embodiments of the method of killing a cell, eg, a T lymphocyte, the extracellular domain of the cell death polypeptide comprises an artificial oligonucleotide sequence. For example, in certain embodiments, the cell death polypeptide comprises an extracellular domain comprising an artificial oligonucleotide sequence. In a specific embodiment, the multimerizing or dimerizing agent is at least one multimerizing or dimerizing optionally comprising a first oligonucleotide and a second oligonucleotide, optionally connected by a linker. An oligonucleotide or comprising said multimerized or dimerized oligonucleotide, wherein said first oligonucleotide and said second oligonucleotide are complementary to said artificial oligonucleotide sequence. In one specific embodiment, the first oligonucleotide and the second oligonucleotide have the same sequence. In a specific embodiment, the first oligonucleotide and the second oligonucleotide are connected in the form of a head and a head or a tail and a tail. In a specific embodiment, when said multimerizing or dimerizing oligonucleotide of said multimerizing agent or dimerizing agent is hybridized to an artificial oligonucleotide sequence of two of said cell death polypeptides: The cell death polypeptide is multimerized or dimerized. In another specific embodiment, an apoptosis induction signal is generated in the cell when the cell death polypeptide is multimerized or dimerized. In a specific embodiment, the cell death polypeptide comprises an intracellular caspase domain, and when the intracellular caspase domain is multimerized or dimerized, an apoptosis induction signal is generated in the cell. In a specific embodiment, the cell is a T lymphocyte.

In certain other embodiments of the method of killing T lymphocytes, the multimerization or dimerization agent is an artificial polypeptide comprising two or more binding domains connected by one or more linkers.

In a specific embodiment, provided herein is a method of killing a cell, eg, a T lymphocyte, wherein the cell comprises a plurality of artificial cell deaths each comprising a caspase or a functional part thereof. A polypeptide, wherein the caspase is caspase 3, caspase 8, or caspase 9 (eg, human caspase 3, caspase 8 or caspase 9), and the cell death polypeptide can be dimerized using an antibody And, when the antibody dimerizes the polypeptide, an apoptosis induction signal is generated in the cell, and the plurality of the plurality is sufficient to dimerize the cell. And D. contacting the artificial polypeptide with an amount of the antibody sufficient to generate a collective apoptosis induction signal sufficient to kill the cell. In a specific embodiment, the cell is a T lymphocyte.

In another specific embodiment, provided herein is a method of killing a cell, eg, a T lymphocyte, wherein the cell comprises a plurality of artificial cells each comprising a caspase or a functional portion thereof. A cell death polypeptide, wherein the caspase is caspase 3, caspase 8 or caspase 9 (e.g. human caspase 3, caspase 8 or caspase 9) and the plurality of artificial polypeptides are each bound to a ligand And / or an extracellular domain comprising a ligand binding portion thereof, wherein the polypeptide is dimerizable with a dimerization agent comprising two of the ligands, and the dimerization agent When the antibody dimerizes two of the polypeptides, an apoptosis-inducing signal is generated in the cells, and a sufficient number of the plurality of artificial cell deaths are sufficient to dimerize the cells. Ripepuchido is dimerized, comprising contacting a sufficient amount of antibody to generate a sufficient collective apoptosis-inducing signal to kill the cells, it is a method. In a specific embodiment, the cell is a T lymphocyte.

In another specific embodiment, provided herein is a method of killing a cell, eg, a T lymphocyte, wherein the cell comprises a plurality of artificial cells each comprising a caspase or a functional portion thereof. A cell death polypeptide, wherein the caspase is caspase 3, caspase 8 or caspase 9 (e.g. human caspase 3, caspase 8 or caspase 9) and the plurality of artificial polypeptides are each a receptor or Comprising an extracellular domain comprising a ligand which binds to its ligand binding portion or its receptor binding portion, said polypeptide being dimerized using a dimerization agent comprising two of said receptors or its ligand binding portion Are characterized in that when the dimerization factor dimerizes two of the polypeptides, an apoptosis induction signal is generated in the cells, The amount of the dimers is sufficient to dimerize a sufficient number of the plurality of artificial polypeptides to dimerize and to generate a collective apoptosis induction signal sufficient to kill the cells. A method comprising contacting with a somatizing agent. In a specific embodiment, the cell is a T lymphocyte.

In another specific embodiment, provided herein is a method of killing a cell, eg, a T lymphocyte, wherein the cell comprises a plurality of artificial cells each comprising a caspase or a functional portion thereof. A cell death polypeptide, wherein said caspase is caspase 3, caspase 8 or caspase 9 (e.g. human caspase 3, caspase 8 or caspase 9), said plurality of artificial polypeptides each being an artificial oligonucleotide A plurality of polypeptides comprising an extracellular domain comprising: and a plurality of said polypeptides being dimerizable with a dimerization factor comprising an oligonucleotide comprising a first oligonucleotide and a second oligonucleotide; One oligonucleotide and the second oligonucleotide have the same nucleotide sequence, the first oligonucleotide and the second oligonucleotide A reotide is optionally connected by a linker, and the first oligonucleotide and the second oligonucleotide are complementary to the artificial oligonucleotide in the extracellular domain of the polypeptide, and the dimeric Somatization factor is 2
When the two cell death polypeptides are dimerized, an apoptosis-inducing signal is generated in the cells, and a sufficient number of the plurality of artificial polypeptides are sufficient to dimerize the cells. And dimerization agent, and contacting with an amount of the dimerization factor sufficient to generate a collective apoptosis induction signal sufficient to kill the cells. In a specific embodiment, the cell is a T lymphocyte.

In a specific embodiment, the T lymphocytes killed according to the methods described herein are CAR-T lymphocytes.

(4.3. Chimeric antigen receptor)
When the cells provided herein are T lymphocytes comprising a cell death polypeptide as described herein, such T lymphocytes, in one embodiment, induce T lymphocytes to an antigen. The chimeric antigen receptor (CAR), which is an artificial membrane-bound protein that stimulates the T lymphocytes to kill cells presenting the antigen, can be included. For example, U.S. Patent No. 7,7 of Eshhar
See No. 41,465. At a minimum, the CAR comprises an antigen, for example, an extracellular domain that binds to an antigen on a cell, a transmembrane domain, and an intracellular that transmits a primary activation signal to an immune cell.
And (cytoplasmic) signaling domain. When all other conditions are met, the CAR is expressed, for example, on the surface of T lymphocytes, and the extracellular domain of the CAR binds to an antigen, the intracellular signaling domain signals T lymphocytes. Transfer to, activate and / or grow,
When the antigen is present on the cell surface, the cells expressing the antigen are killed. T lymphocytes are
Since two signals are required to activate, the primary activation signal and the costimulatory signal, binding of the antigen to the extracellular domain results in the transmission of both the primary activation signal and the costimulatory signal, Usually, CAR also contains a costimulatory domain.

(4.3.1. The intracellular domain of the generic CAR structure)
In one embodiment, the intracellular domain of CAR is an intracellular domain or motif of a protein that is expressed on the surface of T lymphocytes and induces activation and / or proliferation of said T lymphocytes, or Contains domains or motifs. Such domains or motifs can transmit the primary antigen binding signal required for activation of T lymphocytes in response to antigen binding to the extracellular portion of CAR. Usually this domain or motif is IT
It contains AM or is ITAM (immunoreceptor tyrosine-based activation motif). ITAM-containing polypeptides suitable for CAR include, for example, zeta CD3 chain (CD3ζ) or an ITAM-containing portion thereof. In a specific embodiment, the intracellular domain is a CD3ζ intracellular signaling domain. In another specific embodiment, the intracellular domain is derived from a lymphocyte receptor chain, a TCR / CD3 complex protein, an Fc receptor subunit, or an IL-2 receptor subunit.

In certain embodiments, the CAR further comprises one or more costimulatory domains or motifs, eg, as part of the intracellular domain of the polypeptide. The one or more costimulatory domains or motifs may be costimulatory CD27 polypeptide sequences, costimulatory CD28 polypeptide sequences, costimulatory OX40 (C
D134) Polypeptide sequence, costimulatory 4-1BB (CD137) polypeptide sequence, or costimulatory inducible T-cell costimulatory (ICOS) polypeptide sequence, or other costimulatory sequences. It can be or include one or more of a stimulation domain or motif.

The transmembrane domain is any transmembrane domain that can be incorporated into functional CAR, usually CD4.
Alternatively, it can be a transmembrane region derived from a CD8 molecule.

(4.3.2. CAR Transmembrane Domain from CTLA4 or PD-1)
In certain embodiments, the transmembrane domain of CAR, eg, a transmembrane domain from CTLA4 (cytotoxic T lymphocyte antigen 4 or cytotoxic T lymphocyte related protein 4) or PD-1 (programmed death-1) , Are derived from immune system proteins that normally transmit suppression signals to such immune system cells.

In certain embodiments, any of the T lymphocytes provided herein comprising a plurality of cell death polypeptides comprises a transmembrane domain from CTLA4 or PD-1 (programmed cell death 1). In a specific embodiment, T lymphocytes expressing the polypeptide, or any of such polypeptides described herein, are active to proliferate when the polypeptide is bound to the antigen. Or stimulated. In a specific embodiment, the polypeptide induces the T lymphocytes to kill cells expressing the antigen when expressed on the surface of the T lymphocytes.

In specific embodiments of any of the herein described polypeptides wherein the transmembrane domain of the polypeptide is from CTLA4, the CTLA4 transmembrane domain is a mammalian CTLA4, eg, human, primate or Rodents, such as those derived from mouse CTLA4. Preferably, the transmembrane domain does not comprise amino acids from the intracellular domain, the extracellular domain, or either the intracellular or extracellular domain of CTLA4 or PD-1. Specific non-limiting examples of CTLA4 or PD-1 transmembrane domain sequences are provided below.

であるか、又は該アミノ酸配列を含む。別の具体的な実施態様において、CTLA4膜貫通ド
メインは、GenBank受託番号NM_005214.4のヌクレオチド610〜722によってコードされるポ
リペプチド配列であるか、又は該ポリペプチド配列を含む。別の具体的な実施態様におい
て、CTLA4膜貫通ドメインは、アミノ酸配列

であるか、又は該アミノ酸配列を含む。別の具体的な実施態様において、CTLA4膜貫通ド
メインは、GenBank受託番号NM_005214.4のヌクレオチド636〜699によってコードされるポ
リペプチド配列であるか、又は該ポリペプチド配列を含む。別の具体的な実施態様におい
て、CTLA4膜貫通ドメインは、アミノ酸配列

であるか、又は該アミノ酸配列を含む。例えば、Ensemblタンパク質参照番号ENSP0000030
3939.3を参照されたい。別の具体的な実施態様において、CTLA4膜貫通ドメインは、ポリ
ペプチド配列

であるか、又は該ポリペプチド配列を含む。例えば、UNIPROT受託番号P16410を参照され
たい。別の具体的な実施態様において、CTLA4膜貫通ドメインは、ポリペプチド配列

であるか、又は該ポリペプチド配列を含む。例えば、Shinの文献、Blood 119:5678-5687(
2012)を参照されたい。別の具体的な実施態様において、PD-1膜貫通ドメインは、アミノ
酸配列

であるか、又は該アミノ酸配列を含む。Fingerの文献、Gene 197(1-2):177-187(1997)を
参照されたい。別の具体的な実施態様において、PD-1膜貫通ドメインは、アミノ酸配列

であるか、又は該アミノ酸配列を含む。例えば、UNIPROT受託番号Q15116を参照されたい
。別の具体的な実施態様において、PD-1膜貫通ドメインは、アミノ酸配列

であるか、又は該アミノ酸配列を含む。例えば、GenBank受託番号NM_005018.2を参照され
たい。ある実施態様において、本明細書に開示される膜貫通ポリペプチドのうちの1つを
コードするヌクレオチド配列は、配列番号1、配列番号2、配列番号3、配列番号4、配列番
号5、配列番号6、配列番号7、又は配列番号8に開示されるアミノ酸配列のいずれかをコー
ドするヌクレオチド配列を含む。別の具体的な実施態様において、PD-1膜貫通ドメインは
、配列番号1、配列番号2、配列番号3、配列番号4、配列番号5、配列番号6、配列番号7、
もしくは配列番号8に開示される少なくとも10、11、12、13、14、15、16、17、18、19、2
0、もしくは21個の連続するアミノ酸であるか、又は該アミノ酸を含む。ある実施態様に
おいて、本明細書に開示されるポリペプチドのうちの1つをコードするヌクレオチド配列
は、配列番号1、配列番号2、配列番号3、配列番号4、配列番号5、配列番号6、配列番号7
、又は配列番号8に開示される少なくとも10、11、12、13、14、15、16、17、18、19、20
、又は21個の連続するアミノ酸をコードするヌクレオチド配列を含む。該ポリペプチド、
例えば、CARを構築する際、ある実施態様において、ヒト配列を非ヒト配列と組み合わせ
ることができる。例えば、ヒトの細胞外及び細胞内ドメインのアミノ酸配列を含むポリペ
プチド、例えば、CARは、非ヒト種由来の膜貫通ドメインを含むことができ;例えば、マウ
スCTLA4膜貫通ドメイン又はマウスPD-1膜貫通ドメインを含むことができる。より具体的
な実施態様において、該ポリペプチド、例えば、CARは、ヒトの細胞外及び細胞内ドメイ
ンのアミノ酸配列を含み、かつ配列番号5のアミノ酸配列を有するか、又は配列番号5のア
ミノ酸配列からなる膜貫通ドメインを含む。 In a specific embodiment, the CTLA4 transmembrane domain is the polypeptide sequence encoded by exon 3 of the human CTLA4 gene. In another specific embodiment, the CTLA4 transmembrane domain comprises an amino acid sequence

Or includes the amino acid sequence. In another specific embodiment, the CTLA4 transmembrane domain is or comprises the polypeptide sequence encoded by nucleotides 610-722 of GenBank Accession No. NM_005214.4. In another specific embodiment, the CTLA4 transmembrane domain comprises an amino acid sequence

Or includes the amino acid sequence. In another specific embodiment, the CTLA4 transmembrane domain is or comprises the polypeptide sequence encoded by nucleotides 636 to 699 of GenBank Accession No. NM_005214.4. In another specific embodiment, the CTLA4 transmembrane domain comprises an amino acid sequence

Or includes the amino acid sequence. For example, Ensembl protein reference number ENSP0000030
See 3939.3. In another specific embodiment, the CTLA4 transmembrane domain comprises a polypeptide sequence

Or comprises the polypeptide sequence. See, eg, UNIPROT Accession No. P16410. In another specific embodiment, the CTLA4 transmembrane domain comprises a polypeptide sequence

Or comprises the polypeptide sequence. For example, Shin's literature, Blood 119: 5678-5687 (
See 2012). In another specific embodiment, the PD-1 transmembrane domain comprises an amino acid sequence

Or includes the amino acid sequence. See Finger, Gene 197 (1-2): 177-187 (1997). In another specific embodiment, the PD-1 transmembrane domain comprises an amino acid sequence

Or includes the amino acid sequence. See, for example, UNIPROT Accession No. Q15116. In another specific embodiment, the PD-1 transmembrane domain comprises an amino acid sequence

Or includes the amino acid sequence. See, for example, GenBank Accession No. NM_005018.2. In certain embodiments, the nucleotide sequence encoding one of the transmembrane polypeptides disclosed herein comprises: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, a nucleotide sequence encoding any of the amino acid sequences disclosed in SEQ ID NO: 7, or SEQ ID NO: 8. In another specific embodiment, the PD-1 transmembrane domain comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,
Or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 2 disclosed in SEQ ID NO: 8
0 or 21 consecutive amino acids or contain said amino acids. In one embodiment, the nucleotide sequence encoding one of the polypeptides disclosed herein is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7
Or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 disclosed in SEQ ID NO: 8
Or a nucleotide sequence encoding 21 consecutive amino acids. Said polypeptide,
For example, in constructing a CAR, in one embodiment, human sequences can be combined with non-human sequences. For example, a polypeptide comprising the amino acid sequence of human extracellular and intracellular domains, eg, CAR, can comprise a transmembrane domain from a non-human species; eg, a murine CTLA4 transmembrane domain or a murine PD-1 membrane It can contain penetration domains. In a more specific embodiment, the polypeptide, eg, CAR, comprises the amino acid sequence of the extracellular and intracellular domains of human and has the amino acid sequence of SEQ ID NO: 5, or from the amino acid sequence of SEQ ID NO: 5 A transmembrane domain.

(4.3.3. CAR intracellular domain)
The extracellular domain of the polypeptide binds to the antigen of interest. In one embodiment of any of the polypeptides described herein, the extracellular domain comprises a receptor or part of a receptor that binds to the antigen. The extracellular domain can be, for example, a receptor or part of a receptor that binds to the antigen. In one embodiment, the extracellular domain comprises an antibody or antigen binding portion thereof or is an antibody or antigen binding portion thereof. In a specific embodiment, the extracellular domain comprises or is a single chain Fv domain. The single chain Fv domain can include, for example, a V _L linked to V _H by a flexible linker, wherein the V _L and V _H are from an antibody that binds to the antigen .

The antigen to which the extracellular domain of the polypeptide binds may be any antigen of interest, for example an antigen on a tumor cell. The tumor cells can be, for example, cells of solid tumors or cells of hematologic cancer. The antigen may be any tumor type or cancer type cell, such as lymphoma, lung cancer, breast cancer, prostate cancer, adrenocortical carcinoma, thyroid cancer, nasopharyngeal cancer, melanoma, for example, malignant melanoma, skin cancer, colorectal cancer. Cancer, tenoid tumor, fibrogenic small round cell tumor, endocrine tumor, Ewing sarcoma, peripheral anaplastic neuroectodermal tumor, solid germ cell tumor, hepatoblastoma, neuroblastoma, non rhabdomyosarcoma soft tissue sarcoma , Osteosarcoma, retinoblastoma, rhabdomyosarcoma, Wilms tumor,
It can be any antigen expressed on cells such as glioblastoma, myxoma, fibroma, lipoma and the like. In a more specific embodiment, the lymphoma is chronic lymphocytic leukemia (small lymphocytic lymphoma), B cell prolymphocytic leukemia, lymph plasma cell lymphoma, Waldenstrom macroglobulinemia, splenic area Marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B cell lymphoma, MALT lymphoma, nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large cell B cell Lymphoma, mediastinal (thymus) large cell B cell lymphoma, intravascular large cell B cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma, T lymphocyte prolymphocytic leukemia, T lymphocyte large granular lymphocytic Leukemia, invasive NK cell leukemia, adult T lymphocyte leukemia / lymphoma, extranodal NK / T lymphocyte lymphoma, nasal cavity type, enteropathic T lymphocyte lymphoma, hepatosplenic T lymphocyte lymphoma, blastic NK cell Lymphoma, Mycosis fungoides, Sezary syndrome, Primary skin anaplastic large cell lymphoma, Lymphoid papulosis, Angioimmunoblastic T lymphocyte lymphoma, Peripheral T lymphocyte lymphoma (not specified type), Anaplastic large cell lymphoma, Hodgkin's lymphoma,
Or non-Hodgkin's lymphoma.

In a specific embodiment wherein the cancer is chronic lymphocytic leukemia (CLL), the CLL B cells are
It has a normal karyotype. In another specific embodiment, wherein the cancer is chronic lymphocytic leukemia (CLL), the B cells of said CLL carry a 17p deletion, an 11q deletion, a 12q trisomy, a 13q deletion, or a p53 deletion. .

In one embodiment, the antigen is a tumor associated antigen or a tumor specific antigen. In various specific embodiments, but not limited to, the tumor associated antigen or tumor specific antigen is Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) ),
Cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma associated antigen (MAGE), CD19, CD34, CD45, CD99 , CD 117
, Chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), macroscopic cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, dimer form Pyruvate kinase isozyme type M (tumor M2-PK), an abnormal ras protein, or an abnormal p53 protein.

In certain embodiments, the TAA or TSA is a cancer / testis (CT) antigen, such as BAGE, CAGE, CTA.
GE, FATE, GAGE, HCA 661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA 88, NY-ESO-1, NY-SAR-
35, OY-TES-1, SPANXB1, SPA17, SSX, SYCP1, or TPTE.

In certain other embodiments, the TAA or TSA is a carbohydrate or ganglioside, eg,
Fuc-GM1, GM2 (oncofetal antigen-immunogenicity-1; OFA-I-1); GD2 (OFA-I-2), GM3, GD3 and the like.

In certain other embodiments, the TAA or TSA is α-actinin-4, Bage-1, BCR-ABL, Bc.
r-Abl fusion protein, β-catenin, CA 125, CA 15-3 (CA 27.29 \ BCAA), CA 195, CA 24
2, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, CEA, coa-1, dek-can fusion protein, EBNA, EF2, Epstein-Barr virus antigen, ETV6-AML1 fusion protein, HLA-A2, HLA -
A11, hsp70-2, KIAAO 205, Mart2, Mum-1, Mum-2, and Mum-3, neo-PAP, myosin class I
, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, triose phosphate isomerase, Gage 3, 4, 5, 6, 7, GnTV, Herv-K-mel, Lage-1, NA- 88, NY-Eso-1 / Lage-2, SP17, SSX-
2, TRP2-Int2, gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, RAGE
, GAGE-1, GAGE-2, p15 (58), RAGE, SCP-1, Hom / Mel-40, PRAME, p53, H-Ras, HER-2 / n
eu, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, human papilloma virus (HPV) antigens E6 and E7,
TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG
-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, 13-catenin, Mum-1, p16, TAGE, P
SMA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA 225, BTAA, CD68 \ KP1,
CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB 70K
, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD22, CD27, CD3
0, CD70, GD2 (ganglioside G2), EGFRvIII (epithelial growth factor mutant III), sperm protein 1
7 (Sp17), mesothelin, PAP (prostate acid phosphatase), prostain, TARP (T cell receptor gamma alternative reading frame protein), Trp-p8, STEAP1 (prostate 6-transmembrane epithelial antigen 1)
), Abnormal ras protein, or abnormal p53 protein. In another specific embodiment, the tumor associated antigen or tumor specific antigen is the integrin αvβ3 (CD61), galactin (g
alactin), K-Ras (V-Ki-ras2 Carsten rat sarcoma virus oncogene), or Ral-B. Other tumor associated and tumor specific antigens are known to those skilled in the art.

Antibodies and scFv that bind to TSA and TAA, as well as the nucleotide sequences that encode them, are known in the art.

In one specific embodiment, the antigen is not considered TSA or TAA, but is nevertheless an antigen that is associated with tumor cells or damage caused by a tumor.
In certain embodiments, for example, the antigen is, for example, a growth factor, a cytokine, or an interleukin, eg, a growth factor associated with angiogenesis or angiogenesis, a cytokine,
Or an interleukin. As such growth factors, cytokines or interleukins, for example, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), hepatocyte growth factor (HGF) And insulin like growth factor (IGF) or interleukin-8 (IL-8). Tumors can also create a hypoxic environment near the tumor. Thus, in another specific embodiment, the antigen is a hypoxia related factor, such as HIF-1α, HIF-1β, HIF-2α, HIF-2β, HIF-3α, or HIF-3β. Tumors can also cause local damage to normal tissue and cause the release of molecules known as damage associated molecular pattern molecules (DAMP; also known as alarmins). In certain other specific embodiments, therefore, the antigen is DAMP, such as heat shock protein, chromatin related protein high mobility group box 1 (HMGB1), S100A8 (MRP8, calgranulin A), S10.
0A9 (MRP14, calgranulin B), serum amyloid A (SAA), or can be deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate.

In certain embodiments of the polypeptides described herein, the extracellular domain is connected to the transmembrane domain by a linker, spacer, or hinge polypeptide sequence, eg, a sequence derived from CD28 or a sequence derived from CTLA4. ing.

(4.3.4. Dual specificity CAR)
In certain embodiments of the T lymphocytes or methods described herein, the T lymphocytes, in addition to comprising a cell death polypeptide, are divided into two or more polypeptides in which the primary signaling and costimulatory mechanisms are separate. Contains two or more CARs.

In certain embodiments, for example, said T lymphocytes comprise at least two of a cell death polypeptide and
In at least two different polypeptides comprising two different polypeptides, for example a chimeric receptor, an immune signal derived from the binding of a primary signaling polypeptide, for example a chimeric receptor, to a first antigen A costimulatory polypeptide, eg, separated from a costimulatory signal produced by the chimeric receptor, wherein the costimulatory signal is dependent on antigen binding of the second antigen by the second chimeric receptor .

In one embodiment, said T lymphocytes comprise a primary signaling polypeptide comprising a first extracellular antigen binding domain that binds to a first antigen and a first intracellular signaling domain (wherein said primary signal A delivery polypeptide does not comprise a costimulatory domain); and a second extracellular antigen binding domain that binds to a second antigen, or a receptor that binds to said second antigen; a second intracellular signaling domain And a costimulatory polypeptide, wherein said T lymphocyte contains both said first signaling domain and said second signaling domain, respectively, said first antigen and said second Only when activated by the antigen is maximally cytotoxic. In a specific embodiment, binding of said first antigen to said first antigen binding domain without binding of said second antigen to said second binding domain, or first second antigen The binding of the second antigen to the second antigen-binding domain without the binding of the second antigen to the first binding domain is the anergy of the T lymphocyte, or the first antigen of the T-lymphocyte. Or induce non-responsiveness to the second antigen.

In another specific embodiment, said first antigen binding domain and said second antigen binding domain independently comprise an antigen binding portion of a receptor, an antigen binding portion of an antibody, or other peptide-based macromolecules. It is an antigen binding factor. In one specific embodiment, either or both of the first antigen binding domain or the second antigen binding domain is a scFv antibody fragment. In a specific embodiment, either or both of the primary signaling polypeptide or the costimulatory polypeptide further comprises a transmembrane domain. In another specific embodiment, said primary signaling polypeptide or said costimulatory polypeptide comprises a T lymphocyte survival motif. In a specific embodiment, said T lymphocyte survival motif is a CD28 T lymphocyte survival motif. In another specific embodiment, said T lymphocyte survival motif is an IL-7 receptor (IL
-7R) intracellular signaling domain, IL-12 receptor intracellular signaling domain, IL-15
Intracellular signal transduction domain of receptor, intracellular signal transduction domain of IL-21 receptor, or intracellular signal transduction domain of transforming growth factor beta (TGFβ) receptor. In another more specific embodiment, said primary signaling polypeptide or said costimulatory polypeptide comprises a portion of a CD28 molecule comprising a T lymphocyte survival motif. In a more specific embodiment, said primary signaling polypeptide or said costimulatory polypeptide comprises a CD28 molecule comprising a T lymphocyte survival motif. In one specific embodiment, the first intracellular signaling domain comprises a polypeptide sequence comprising an immunoreceptor tyrosine-based activation motif (ITAM). In a more specific embodiment, the polypeptide sequence is a CD3ζ signaling domain.

In one specific embodiment, the first antigen is an antigen on a tumor cell. In a more specific embodiment, the tumor cells are cells of a solid tumor. In another more specific embodiment, the tumor cells are hematologic cancer cells. In another specific embodiment, the antigen is a tumor associated antigen or a tumor specific antigen. In a more specific embodiment, said tumor associated antigen or tumor specific antigen is Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AF)
P), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA 19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma related antigen (MAGE), CD34,
CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), macroscopic cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A (recognized by T lymphocytes) Melanoma antigen to be detected; MART-1), myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, double dose Body form of pyruvate kinase isoenzyme type M2 (tumor M2-PK), abnormal ras protein, or abnormal p53
It is a protein.

In another specific embodiment, the second antigen is a growth factor, a cytokine, or an interleukin. The second antigen is a growth factor, a cytokine, or an interleukin associated with angiogenesis or angiogenesis. In a more specific embodiment, said second antigen is vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet derived growth factor (P
DGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), or interleukin-8 (IL-
8).

In another specific embodiment, the signaling activation provided by the second antigen is non-antigenic but associated with hypoxia. In more specific embodiments, the stimulation is hypoxia inducible factor-1 alpha (HIF-1 alpha), HIF-1 beta, HIF-2 alpha, HIF-2 beta, HIF-3 alpha, or HIF-3 beta.
Induced by the activation of

  In another specific embodiment, the second antigen is an interleukin.

In another specific embodiment, the second antigen is a damage associated molecular pattern molecule (DAMP; also known as alarmin). In a more specific embodiment, said DAMP is a heat shock protein, chromatin related protein high mobility group box 1 (HMGB1), S100A8 (aka MRP8 or calgranulin A), S100A9 (aka MRP14 or cal) Granulin B), serum amyloid
A (SAA), deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate
fate).

In one specific embodiment, the second antigen is an antigen on an antibody that binds to an antigen presented by tumor cells.

In specific embodiments of any of the embodiments herein, the costimulatory polypeptide comprises one or more costimulatory domains. In a specific embodiment, the one or more costimulatory domains comprise a costimulatory CD27 polypeptide sequence, a costimulatory CD28 polypeptide sequence, a costimulatory OX40 (CD134).
It comprises one or more of a polypeptide sequence, a costimulatory 4-1BB (CD137) polypeptide sequence, or a costimulatory inducible T cell costimulatory (ICOS) polypeptide sequence.

In a specific embodiment, said primary signaling polypeptide comprises an extracellular tumor antigen binding domain and a CD3ζ signaling domain, wherein said costimulatory polypeptide is
An antigen binding domain (wherein the antigen is an angiogenic or angiogenic factor) and one or more costimulatory molecule signaling domains. The angiogenic factor can be, for example, VEGF. The one or more costimulatory molecule signaling motifs can include, for example, costimulatory signaling domains from each of CD28, OX40, and 4-1BB. In a more specific embodiment, said primary signaling polypeptide comprises an extracellular tumor antigen binding domain and a CD3ζ.
A signal transduction domain, wherein the costimulatory polypeptide comprises an antigen binding domain
Here, the antigen is VEGF) and costimulatory signaling domains from each of CD28, OX40, and 4-1BB.

In a more specific embodiment, said primary signaling polypeptide or said costimulatory polypeptide comprises a T lymphocyte survival motif. In a more specific embodiment, said T lymphocyte survival motif is an intracellular signaling domain of IL-7 receptor (IL-7R), an intracellular signaling domain of IL-12 receptor, an IL-15 receptor Or the intracellular signaling domain of the IL-21 receptor, or the intracellular signaling domain of the transforming growth factor beta (TGF beta) receptor, or derived from these intracellular signaling domains Do. In a more specific embodiment of the T-lymphocytes, therefore, the primary signaling polypeptide comprises an extracellular tumor antigen binding domain and a CD3ζ signaling domain, in which case:
The costimulatory polypeptide comprises an antigen binding domain (wherein the antigen is VEGF), an IL-7 receptor intracellular T lymphocyte survival motif, and co-derived from each of CD28, OX40, and 4-1BB. It contains a stimulation signal transduction domain.

In another specific embodiment of said T lymphocytes, said first antigen is a tumor specific antigen or a tumor associated antigen, said first intracellular signaling domain comprises a CD3ζ signaling domain; In that case, the costimulatory polypeptide comprises an antigen binding domain that binds to the second antigen, and costimulatory signaling domains from each of CD28, OX40, and 4-1BB. In a more specific embodiment, the costimulatory polypeptide comprises an intracellular T lymphocyte survival motif, eg, an intracellular signaling domain of an IL-7 receptor (IL-7R), an intracellular of an IL-12 receptor. A signaling domain, an intracellular signaling domain of an IL-15 receptor, an intracellular signaling domain of an IL-21 receptor, or an intracellular signaling domain of a transforming growth factor beta (TGF beta) receptor, or It further comprises T lymphocyte survival motifs derived from these intracellular signaling domains.

In specific embodiments of any of the T lymphocytes provided herein, the second antigen is VEGF or IL-4.

In another aspect, provided herein is a cell death polypeptide, a costimulatory poly comprising a first extracellular antigen binding domain that binds a first antigen and a first intracellular signaling domain. And a second extracellular antigen-binding domain that binds to a second antigen, or a receptor that binds to the second antigen; and a second signal transduction domain; In which the primary signaling polypeptide is a T lymphocyte containing a costimulatory domain); wherein the modified lymphocyte comprises the first signaling domain and the second signaling domain. Both domains are maximally cytotoxic only when activated by the first and second antigens, respectively. In a specific embodiment, binding of said first antigen to said first antigen binding domain without binding of said second antigen to said second binding domain, or first second antigen The binding of the second antigen to the second antigen binding domain without the binding of the second antigen to the first binding domain is the anergy of the T lymphocyte, or of the T lymphocyte to the first antigen. Induce non-responsiveness. In a specific embodiment, said first antigen binding domain and said antigen binding domain are independently an antigen binding portion of a receptor or an antigen binding portion of an antibody. In another specific embodiment, either or both of said first antigen binding domain or said second antigen binding domain are scFv antibody fragments. In a specific embodiment, the costimulatory polypeptide and / or the primary signaling polypeptide further comprises a transmembrane domain. In more specific embodiments, the costimulatory polypeptide or the primary signaling polypeptide comprises a T lymphocyte survival motif, such as any of the T lymphocyte survival motifs described herein. In another specific embodiment, the first antigen is an antigen on a tumor cell, such as a solid tumor cell or a hematologic cancer cell.
In a specific embodiment, the first antigen is a tumor associated antigen or a tumor specific antigen such as Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), Cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma associated antigen (MAGE), CD34, CD45, CD99, CD117 , Chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), macroscopic cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, pyruvate in dimeric form Kinase A Sozaimu type M2 (tumor M2-PK), abnormal ras proteins, abnormal p53 protein, CD19, CD22, CD27, C
D30, CD70, GD2 (ganglioside G2), EGFRvIII (epithelial growth factor mutant III), sperm protein 17 (Sp17), mesothelin, PAP (prostate acid phosphatase), prostain, TARP (T cell receptor γ alternative reading frame protein ), Trp-p8, or STEAP1 (6-transmembrane epithelial antigen 1 of prostate). In another specific embodiment, the tumor associated antigen or tumor specific antigen is integrin αvβ3 (CD61), galactin (galactin), K-Ras (V-Ki-ras2 karsten rat sarcoma virus oncogene), or Ral -B.

In one specific embodiment, the second intracellular signaling domain comprises a polypeptide sequence comprising an immunoreceptor tyrosine-based activation motif (ITAM), eg, a CD3ζ signaling domain. In a specific embodiment, the second antigen is a growth factor, a cytokine, or an interleukin. In another specific embodiment, said second antigen is a growth factor, cytokine or interleukin associated with angiogenesis or angiogenesis, eg VEGF, bFGF, PDGF, HGF, IGF or IL-8 It is. In another more specific embodiment, signaling by said second chimeric receptor is hypoxia related factors such as HIF-1α, H.
It is induced by activation of IF-1β, HIF-2α, HIF-2β, HIF-3α, or HIF-3β. In another specific embodiment, the second antigen is an interleukin. In another specific embodiment, said second antigen is DAMP, eg heat shock protein, HMGB1, S100A8.
, S100A9, SAA, DNA, ATP, uric acid, or heparin sulfate. In another specific embodiment, the second antigen is a peptide to be administered, such as an antibody or a synthetic polypeptide. In another specific embodiment, the second antigen is an antigen on an antibody that binds to an antigen presented by a tumor cell. In one specific embodiment, the costimulatory polypeptide comprises one or more costimulatory domains, such as a costimulatory CD27 polypeptide sequence, a costimulatory CD28 polypeptide sequence, a costimulatory OX40 (CD134) polypeptide sequence, a costimulatory polypeptide. 4-1BB (CD 137)
The polypeptide sequence or one or more of a costimulatory inducible T cell costimulatory (ICOS) polypeptide sequence. In any of the above embodiments, in a specific embodiment, the costimulatory polypeptide or the primary signaling polypeptide comprises a T lymphocyte survival motif, eg, the T lymphocyte survival motif is an IL. -7 receptor (IL-7R) intracellular signaling domain, IL-12 receptor intracellular signaling domain, IL-15 receptor intracellular signaling domain, IL-21 receptor intracellular signaling Domain, or transforming growth factor β (TGFβ)
It is the intracellular signaling domain of the receptor or is derived from these intracellular signaling domains.

(4.4. Isolated polypeptide)
Any of the polypeptides provided herein, including the CTLA4 or PD-1 transmembrane domain, can be, for example, acylated, amidated, glycosylated, methylated, phosphorylated, sulfated, sumo
And ubiquitination can be modified. The polypeptide can be labeled with a label capable of providing a detectable signal, for example, radioactive isotopes and fluorescent compounds. One or more side chains of the first or second polypeptide can be derivatized, for example, derivatization of a lysinyl residue and an amino terminal residue with succinic acid or another carboxylic acid anhydride, or, for example, Imido esters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;
-Methylisourea; 2,4 pentanedione; and derivatization by reaction with glyoxylate catalyzed by transaminase. The carboxyl side group aspartyl or glutamyl is a carbodiimide (RN = C = N-R '), for example, 1-cyclohexyl-3- (2-morpholinyl- (4-ethyl) carbodiimide or 1-ethyl-3- (4-) It can be selectively modified by reaction with azonia-4,4-dimethylpentyl) carbodiimide.

(4.5. Isolated nucleic acid)
The polypeptides (eg, chimeric receptors) described herein can be encoded by a polynucleotide sequence according to methods well known in the art. The polynucleotide can be included in any polynucleotide vector suitable for transformation of immune cells, eg, T lymphocytes. For example, T lymphocytes may comprise synthetic, lentiviral or retroviral vectors, autonomously replicating plasmids, viruses (e.g. retroviruses) comprising a polynucleotide encoding a first and a second polypeptide (e.g. a chimeric receptor) , Lentivirus, adenovirus, or herpes virus), etc. can be used. Suitable lentiviral vectors for transformation of T lymphocytes include, for example, US Pat. Nos. 5,994,136; 6,165, the disclosure of which is fully incorporated herein by reference.
And lentiviral vectors described in U.S. Pat. No. 782, U.S. Pat. No. 6,428,953; U.S. Pat. No. 7,083,981; and U.S. Pat. No. 7,250,299, but not limited thereto. Suitable HIV vectors for transformation of T lymphocytes include, but are not limited to, for example, the vectors described in US Pat. No. 5,665,577, the disclosure of which is fully incorporated herein by reference. .

For example, nucleic acids useful in the production of first and second polypeptides in T lymphocytes include DNA, RNA, or nucleic acid analogs. Nucleic acid analogues include a base moiety, a sugar moiety,
Or can be modified in the phosphate ester backbone and can include deoxyuridine substitution for deoxythymidine, 5-methyl-2'-deoxycytidine or 5-bromo-2'-deoxycytidine substitution for deoxycytidine . Modification of the sugar moiety is 2'-O-methyl or 2'-
Modification of the 2 'hydroxyl of a ribose sugar to form an O-allyl sugar can be included.
The deoxyribose phosphate ester backbone is a morpholino nucleic acid in which each base moiety is bound to a 6-membered morpholino ring, or a deoxyphosphate backbone is substituted by a pseudopeptide backbone to form a peptide nucleic acid in which four bases are retained It can be modified to For example, Summerton and Weller (1997), Antisense Nucleic Acid Drug Dev. 7:
187-195; and Hyrup et al. (1996), Bioorgan. Med. Chain. 4: 5-23. In addition, the deoxyphosphate ester backbone can be substituted, for example, with a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.

(4.6. Cell)
Non-limiting examples of cells that may use cell death polypeptides and multimerization or dimerization factors include natural killer (NK) cells, dendritic cells (DC), placental stem cells (eg, their disclosure Placental stem cells disclosed in US Patent Nos. 7,468, 276; 8,057, 788, and 8, 202, 703, which are fully incorporated herein by reference), umbilical cord blood, placental blood, peripheral blood, bone marrow, dental pulp,
Mesenchymal-like stem cells derived from adipose tissue, osteochondral tissue and the like; embryonic stem cells, embryonic germ cells, neural crest stem cells, neural stem cells, and differentiated cells (for example, fibroblasts and the like) I will not. The cell death polypeptide and multimerization or dimerization factor can also be used in tumor cell lines, for example for the purpose of animal model experiments.

In a specific embodiment, the cells comprising the polypeptide provided herein are T lymphocytes. T lymphocytes comprising a polypeptide provided herein may be naive T lymphocytes or MHC restricted T lymphocytes. In one embodiment, the T lymphocytes are tumor infiltrating lymphocytes (TIL). In one embodiment, the T lymphocytes are isolated from tumor biopsies or expanded from T lymphocytes isolated from tumor biopsies. In certain other embodiments, the T lymphocytes are isolated from peripheral blood, umbilical cord blood, or lymph, or expanded from T lymphocytes expanded from peripheral blood, umbilical cord blood, or lymph. It is

Preferably, the immune cells, eg, T lymphocytes, used in the method are autologous to the individual to whom the T lymphocytes are to be administered. In certain other embodiments, the T lymphocytes are allogeneic to the individual to whom the T lymphocytes are to be administered. Graft versus host disease (GVHD) in an individual when preparing T lymphocytes using allogeneic T lymphocytes
It is preferred to select T lymphocytes which reduce the likelihood of For example, in one embodiment, virus specific T lymphocytes are selected for the preparation of T lymphocytes; such lymphocytes
It is believed that the natural ability to bind to any recipient antigen and as a result be activated by the antigen is greatly diminished. In certain embodiments, rejection of allogeneic T lymphocytes by the recipient is alleviated by co-administration of one or more immunosuppressants, such as cyclosporin, tacrolimus, sirolimus, cyclophosphamide, etc., to the host. it can.

In one embodiment, T lymphocytes are obtained from an individual and optionally subsequently expanded, followed by a polynucleotide encoding a cell death polypeptide, and optionally one or more CARs 1
Transform with the above polynucleotides and optionally later expand. In another embodiment,
T lymphocytes are obtained from the individual, optionally subsequently expanded, and then transformed with a polynucleotide encoding a cell death polypeptide, and optionally one or more polynucleotides encoding one or more CARs, optionally subsequently Expanding. One cell containing any of the polynucleotides is
Selection is possible using the above selectable markers.

In certain embodiments, any of the T lymphocytes provided herein is CTLA4 or PD-1.
In addition to transmembrane domain-containing polypeptides, they express or include natural TCR proteins capable of forming natural TCR complexes, such as, for example, TCR-α and TCR-β. In certain other embodiments, either or both of the TCR-α and TCR-β-encoding native genes of the T lymphocyte are modified to be nonfunctional, eg, deleted in part or all. Insert a mutation, etc.

In certain embodiments, any of the T lymphocytes provided herein are isolated from a tumor lesion, eg, tumor infiltrating lymphocytes; such T lymphocytes are specific for TSA or TAA. It is considered to be.

T lymphocytes, and T lymphocytes comprising a polypeptide comprising a CD3ζ signaling domain and a CD28 costimulatory domain, are expanded with antibodies to CD3 and CD28, eg, beads or antibodies attached to the surface of a cell culture plate For example, U.S. Patent No. 5,948,893
Nos. 6,534,055; 6,352,694; 6,692,964; 6,887,466; and 6,905,681.

In any of the above embodiments, the antigen and / or antibody may be present free in the medium in which T lymphocytes are cultured, or either or both, on a solid support, eg Can be attached to tissue culture plastic surfaces, beads, etc.

The T lymphocytes provided herein can optionally include a second type of "suicide gene" or "safety switch" in addition to the cell death polypeptide. For example, the T lymphocytes may
In one embodiment, HSV causes T lymphocyte death when contacted with ganciclovir
The thymidine kinase gene (HSV-TK) can be included. In another embodiment, the T lymphocyte is a human that allows inducible caspases, such as inducible caspase 9 (i-caspase 9), for example, dimerization with caspase 9 and certain small molecule drugs. Or express fusion proteins with the FK506 binding protein. Straathof et al., Blood 105 (11): 4
See, eg, H.247-4254 (2005).

(4.7. Method of using cells containing cell death polypeptide)
The cells provided herein, including cell death polypeptides and optionally one or more of the CARs described elsewhere herein, eg, using T lymphocytes, are described herein. An individual having one or more types of cells that it is desired to be targeted by cells, eg, one or more types of cells to be killed, can be treated. In one embodiment, the cell to be killed is a cancer cell, eg a tumor cell. In a specific embodiment, the cancer cell is a solid tumor cell. In a specific embodiment, the cell is a lymphoma, a lung cancer,
Breast cancer, prostate cancer, adrenocortical cancer, thyroid cancer, nasopharyngeal cancer, melanoma, for example, malignant melanoma, skin cancer, colorectal cancer, tendonoma, fibrogenic small round cell tumor, endocrine tumor, Ewing sarcoma, Peripheral anaplastic neuroectodermal tumor, solid germ cell tumor, hepatoblastoma, neuroblastoma, non rhabdomyosarcoma soft tissue sarcoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, Wilms tumor, glioblastoma, It is a cell such as myxoma, fibroma and lipoma. In a more specific embodiment, the lymphoma is chronic lymphocytic leukemia (small lymphocytic lymphoma), B cell prolymphocytic leukemia, lymph plasma cell lymphoma, Waldenstrom macroglobulinemia, splenic area Marginal zone lymphoma, plasma cell myeloma,
Plasmacytoma, extranodal marginal zone B cell lymphoma, MALT lymphoma, nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymus) large cell B cell lymphoma, intravascular large cell B cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma, T lymphocyte prelymphocytic leukemia, T lymphocyte large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T lymphocyte leukemia / lymphoma, extranodal NK / T lymphocyte lymphoma, nasal type,
Enteropathy type T lymphocyte lymphoma, hepatosplenic T lymphocyte lymphoma, blastic NK cell lymphoma, mycosis fungosarcoma, Sezary's syndrome, cutaneous primary anaplastic large cell lymphoma, lymphomatous papulosis, hemangioimmunoblastic
It may be T lymphocyte lymphoma, peripheral T lymphocyte lymphoma (unspecified type), anaplastic large cell lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

In certain embodiments, the modified cells described herein, eg, modified T lymphocytes, when administered to a subject in need thereof, select multimerizing agents and cell death polypeptides In the combination of patients, the patient population, for example, a patient population to which T lymphocytes have been administered (eg,
Or subpopulations). By way of example only, where the multimerization factor selected is the antibody rituximab, in certain embodiments, the patient population is a B-cell cancer,
For example, an individual with B cell lymphoma.

An individual having a disease or disorder treatable by such cells, eg, T lymphocytes,
For example, it is known to those skilled in the art that the efficacy of the cells, eg, T lymphocytes, after administration to an individual having cancer, is indicative of the progression of the disease or disorder specific to the particular disease or disorder. It can be assessed by one or more criteria being Generally, administration of the cells to such individuals is such that one or more of the criteria are detectable from or towards the value or range of the disease state or towards the normal value or range thereof. , For example, when transitioning significantly.

The cells, eg, T lymphocytes, can be any pharmaceutically acceptable solution, preferably a solution suitable for delivery of live cells, eg, saline (eg, Ringer's solution), gelatin, carbohydrates
It can be formulated in (for example, lactose, amylose, starch and the like), fatty acid ester, hydroxymethylcellulose, polyvinyl pyrrolidine and the like. Such preparations are preferably sterilized prior to addition to the cells, and also include lubricants, preservatives, stabilizers, emulsifiers, salts affecting the osmotic pressure, buffers, coloring agents, etc. Can be mixed with the Pharmaceutical carriers suitable for use in formulating the cells are known in the art and are described, for example, in WO 96/05309.

In certain embodiments, the cells, eg, T lymphocytes, are formulated into individual doses, wherein the individual doses are at least, at most, or about 1 × 10 ⁴ , 5 × 10 ⁴ , 1 × 10 ⁴ . ⁵ , 5 × 10 ⁵ , 1
× 10 ⁶ , 5 × 10 ⁶ , 1 × 10 ⁷ , 5 × 10 ⁷ , 1 × 10 ⁸ , 5 × 10 ⁸ , 1 × 10 ⁹ , 5 × 10 ⁹ , 1 × 10 ¹⁰ , 5 × 10 ¹⁰
Or 1 × 10 ¹¹ T lymphocytes. In certain embodiments, the cells are formulated for intravenous, intraarterial, parenteral, intramuscular, subcutaneous, intrathecal or intraocular administration, or for administration into a particular organ or tissue.

(5. Example)
(5.1. Example 1: Treatment of B cell lymphoma)
The individual is presenting symptoms of B cell lymphoma of B cell chronic lymphocytic leukemia. Examination of B cells from the individual reveals that the B cells carry a 17p deletion. T lymphocytes are obtained from the individual and transfected with a lentiviral vector comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), and an extracellular domain comprising a mimotope which can be bound by the antibody rituximab, and a caspase A second lentiviral vector comprising a nucleotide sequence encoding a dimerizable cell death polypeptide comprising an intracellular domain comprising 9 domains is transfected. The T lymphocytes are expanded to a number sufficient for dosing with CD3 + CD28-coated beads. The chimeric receptor comprises: an extracellular antigen binding region that binds to CD19; a transmembrane domain from CTLA4; an intracellular costimulatory domain from CD28; and an intracellular CD3
Contains the vaginal domain. The individual is administered 10 ⁹ to 10 ¹⁰ T lymphocytes in 200 mL of saline solution by intravenous infusion over 30 minutes. The individual is monitored for two weeks thereafter to establish a reduction of at least 90% of CD19 + B cells in the blood of the individual. If, after administration of T lymphocytes, the patient shows that he or she seems to suffer due to the T lymphocytes (for example, dyspnea, fever, abnormal serum cytokine levels, rash, etc.), 200 to 500 mg / m ² Rituximab is administered at a dosage or until symptoms are alleviated.

5.2. Example 2: Treatment of B cell lymphoma
The individual is presenting symptoms of B cell lymphoma of B cell chronic lymphocytic leukemia. Examination of B cells from the individual reveals that the B cells carry a 17p deletion. About 10 ⁶ T lymphocytes are obtained from the individual and encode a cell death polypeptide comprising an extracellular domain comprising a mimotope which may be bound by the antibody rituximab and an intracellular domain comprising a caspase 8 domain. A lentiviral vector containing the nucleotide sequence is transfected and a lentiviral vector containing the nucleotide sequence encoding CAR is transfected. The CAR has an extracellular antigen binding region that binds to CD19; a transmembrane domain derived from PD-1; CD2
8 and an intracellular costimulatory domain; and an intracellular CD3ζ domain. T lymphocytes expressing CAR are administered to the individual without prior expansion of the T lymphocytes. The individual is administered 10 ⁵ to 10 ⁶ T lymphocytes in 200 mL of saline solution by intravenous infusion over 30 minutes. The individual is monitored for two weeks thereafter to establish a reduction of at least 90% of CD19 + B cells in the blood of the individual. If, after administration of T lymphocytes, the patient shows that he or she seems to suffer due to the T lymphocytes (for example, dyspnea, fever, abnormal serum cytokine levels, rash, etc.), 200 to 500 mg / m ² Rituximab is administered at a dosage or until symptoms are alleviated.

(5.3. Example 3: Treatment of B cell lymphoma)
The individual is presenting symptoms of B cell lymphoma of B cell chronic lymphocytic leukemia. Examination of B cells from the individual reveals that the B cells carry a p53 deletion. T lymphocytes are obtained from the individual, and the wrench contains a nucleotide sequence encoding a cell death polypeptide comprising an extracellular domain comprising an epitope that can be bound by the antibody rituximab and an intracellular domain comprising a caspase 3 domain. The viral vector is transfected and the lentiviral vector containing the nucleotide sequence encoding CAR is transfected. Said
T lymphocytes are expanded to a number sufficient for dosing with CD3 + CD28-coated beads. Said
CAR has an extracellular antigen binding region that binds to CD19; a transmembrane domain derived from CTLA4; CD28, 4-1BB
And an intracellular costimulatory domain derived from each of OX40; and an intracellular CD3ζ domain.
The individual is administered 10 ⁹ to 10 ¹⁰ T lymphocytes in 200 mL of saline solution by intravenous infusion over 30 minutes. The individual is monitored for two weeks thereafter to establish a reduction of at least 90% of CD19 + B cells in the blood of the individual. After administration of T lymphocytes, the patient
If lymphocytes show signs of distressing (eg dyspnea, fever, abnormal serum cytokine levels, rash etc), at a dose of 200 to 500 mg / m ² or until the symptoms are alleviated
Administer rituximab.

5.4. Example 4: Treatment of B-cell Lymphoma
The individual is presenting symptoms of B cell lymphoma of B cell chronic lymphocytic leukemia. Examination of B cells from the individual reveals that the B cells carry a p53 deletion. About 10 ⁶ T lymphocytes are obtained from the individual and encode a cell death polypeptide comprising an extracellular domain comprising an epitope which can be bound by the antibody rituximab and an intracellular domain comprising a caspase 9 domain A lentiviral vector containing the nucleotide sequence is transfected and a lentiviral vector containing the nucleotide sequence encoding CAR is transfected. The CAR has an extracellular antigen binding region that binds to CD19; a transmembrane domain derived from PD-1; CD2
8, 4-1BB, and an intracellular costimulatory domain derived from each of OX40; and an intracellular CD3ζ domain. T lymphocytes expressing CAR are administered to the individual without prior expansion of the T lymphocytes. The individual is administered 10 ⁵ to 10 ⁶ T lymphocytes in 200 mL of saline solution by intravenous infusion over 30 minutes. The individual is monitored for two weeks thereafter to establish a reduction of at least 90% of CD19 + B cells in the blood of the individual. If, after administration of T lymphocytes, the patient shows that he or she seems to suffer due to the T lymphocytes (for example, dyspnea, fever, abnormal serum cytokine levels, rash, etc.), 200 to 500 mg / m ² Rituximab is administered at a dosage or until symptoms are alleviated.

5.5. Example 5: Treatment of Prostate Cancer
The individual presents with symptoms of stage T2 prostate cancer that has not spread to regional or other lymph nodes (N0, M0). Histological grade is known as G2. Overall, the individual is found to have stage II prostate cancer. The individual is treated with CAR in 200 mL of saline solution.
10 < ⁹ > -10 < ¹⁰ > T-lymphocytes, including <RTIgt; I, </ RTI> are administered by intravenous infusion over 30 minutes. The CAR
Contains an extracellular antigen binding region that binds PSCA, a transmembrane domain from CTLA4, an intracellular costimulatory domain from CD28, and an intracellular CD3ζ domain. The T lymphocyte comprises an extracellular domain comprising an epitope which can be bound by the antibody rituximab, caspase 3, caspase 8,
Or a cell death polypeptide comprising an intracellular domain comprising a caspase 9 domain. The individual is reassessed for stage of prostate cancer and spread to lymph nodes and histologic examination of biopsied prostate tissue is performed 30, 60 and 90 days after administration. If, after administration of T lymphocytes, the patient shows that he or she seems to suffer due to the T lymphocytes (for example, dyspnea, fever, abnormal serum cytokine levels, rash, etc.), 200 to 500 mg / m ² Rituximab is administered at a dosage or until symptoms are alleviated.

5.6. Example 6: Treatment of Prostate Cancer
The individual presents with symptoms of stage T2 prostate cancer that has not spread to regional or other lymph nodes (N0, M0). Histological grade is known as G2. Overall, the individual is found to have stage II prostate cancer. The individual is treated with CAR in 200 mL of saline solution.
10 < ⁹ > -10 < ¹⁰ > T-lymphocytes, including <RTIgt; I, </ RTI> are administered by intravenous infusion over 30 minutes. The CAR
Contains an extracellular antigen binding region that binds PSCA, a transmembrane domain from PD-1, an intracellular costimulatory domain from CD28, and an intracellular CD3ζ domain. The T lymphocytes also comprise a cell death polypeptide comprising an extracellular domain comprising an epitope which can be bound by the antibody rituximab, and an intracellular domain comprising a caspase 3, caspase 8 or caspase 9 domain. The individual is reassessed for stage of prostate cancer and spread to lymph nodes and histologic examination of biopsied prostate tissue is performed 30, 60 and 90 days after administration. If, after administration of T lymphocytes, the patient shows that he or she seems to suffer due to the T lymphocytes (for example, dyspnea, fever, abnormal serum cytokine levels, rash, etc.), 200 to 500 mg / m ² Rituximab is administered at a dosage or until symptoms are alleviated.

(5.7. Example 7: Treatment of Prostate Cancer)
The individual presents with symptoms of stage T2 prostate cancer that has not spread to regional or other lymph nodes (N0, M0). Histological grade is known as G2. Overall, the individual is found to have stage II prostate cancer. The individual is treated with CAR in 200 mL of saline solution.
10 < ⁹ > -10 < ¹⁰ > T-lymphocytes, including <RTIgt; I, </ RTI> are administered by intravenous infusion over 30 minutes. The CAR
Is an extracellular antigen binding region that binds PSCA, a transmembrane domain derived from CTLA-4, CD28, 4-1BB,
And an intracellular costimulatory domain derived from each of OX40, as well as an intracellular CD3ζ domain.
The T lymphocytes also comprise a cell death polypeptide comprising an extracellular domain comprising an epitope which can be bound by the antibody rituximab, and an intracellular domain comprising a caspase 3, caspase 8 or caspase 9 domain. The individual is reassessed for stage of prostate cancer and spread to lymph nodes and histologic examination of biopsied prostate tissue is performed 30, 60 and 90 days after administration. If, after administration of T lymphocytes, the patient shows that he or she seems to suffer due to the T lymphocytes (eg, dyspnea, fever, abnormal serum cytokine levels, rash, etc.), 200 to 500 mg / m ²
Rituximab is administered at a dosage of or until symptoms are alleviated.

5.8. Example 8 Treatment of Prostate Cancer
The individual presents with symptoms of stage T2 prostate cancer that has not spread to regional or other lymph nodes (N0, M0). Histological grade is known as G2. Overall, the individual is found to have stage II prostate cancer. The individual is treated with CAR in 200 mL of saline solution.
10 < ⁹ > -10 < ¹⁰ > T-lymphocytes, including <RTIgt; I, </ RTI> are administered by intravenous infusion over 30 minutes. The CAR
Contains an extracellular antigen binding region that binds PSCA, a transmembrane domain from PD-1, an intracellular costimulatory domain from each of CD28, 4-1BB, and OX40, and an intracellular CD3ζ domain. The T
Lymphocytes also comprise a cell death polypeptide comprising an extracellular domain comprising an epitope which can be bound by the antibody rituximab and an intracellular domain comprising a caspase 3, caspase 8 or caspase 9 domain. The individual is reassessed for stage of prostate cancer and spread to lymph nodes and histologic examination of biopsied prostate tissue is performed 30, 60 and 90 days after administration. If, after administration of T lymphocytes, the patient shows that he or she seems to suffer due to the T lymphocytes (for example, dyspnea, fever, abnormal serum cytokine levels, rash, etc.), 200 to 500 mg / m ² Rituximab is administered at a dosage or until symptoms are alleviated.

(Equivalent)
The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the subject matter provided herein, in addition to those described, will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Various publications, patents, and patent applications are cited herein, the disclosures of which are incorporated by reference in their entirety.

Various publications, patents, and patent applications are cited herein, the disclosures of which are incorporated by reference in their entirety.
The present application provides the invention of the following configuration.
(Configuration 1)
A T lymphocyte comprising an artificial cell death polypeptide comprising an apoptosis induction domain, wherein the artificial cell death polypeptide comprises an extracellular domain comprising a CD20 epitope, a transmembrane domain, and the apoptosis induction domain And the apoptosis-inducing domain is caspase 3, caspase 8 or caspase 9, and the polypeptide is dimerizable using an anti-CD20 antibody that binds to the CD20 epitope. And said T lymphocytes, wherein an apoptosis induction signal is generated in said T lymphocytes when said antibody dimerizes said polypeptide.
(Configuration 2)
The T lymphocyte of configuration 1, wherein said antibody is approved by the US Food and Drug Administration for any use.
(Configuration 3)
Whether the antibody is rituximab and the extracellular domain comprises a CD20 epitope to which the rituximab binds;
Whether the antibody is tosituzumab and the extracellular domain comprises a CD20 epitope to which tosituzumab binds;
The antibody is ibritumomab, and the extracellular domain comprises a CD20 epitope to which the ibritumomab binds; or
The T lymphocyte according to configuration 1, wherein said antibody is ofatumumab and said extracellular domain comprises a CD20 epitope to which said ofatumumab binds.
(Configuration 4)
A pharmaceutical composition for killing T lymphocytes, wherein the T lymphocytes comprise a plurality of artificial cell death polypeptides each comprising an apoptosis induction domain, wherein the artificial cell death polypeptide comprises a CD20 epitope. A transmembrane protein comprising an exodomain, a transmembrane domain, and an intracellular domain comprising said apoptosis induction domain, said apoptosis induction domain is caspase 3, caspase 8 or caspase 9, said polypeptide is It is characterized that it can be dimerized using an anti-CD20 antibody that binds to a CD20 epitope, and that when the antibody dimerizes the polypeptide, an apoptosis induction signal is generated in the T lymphocytes. And wherein the plurality of artificial cell death polypeptides are dimerized and the collective apoptosis induction signal is sufficient to kill the T lymphocytes. Said pharmaceutical composition comprising an amount of said antibody sufficient to generate.
(Configuration 5)
5. The pharmaceutical composition according to configuration 4, wherein said antibody is approved by the U.S. Food and Drug Administration for any use.
(Configuration 6)
Whether the antibody is rituximab and the extracellular domain comprises a CD20 epitope to which the rituximab binds;
Whether the antibody is tosituzumab and the extracellular domain comprises a CD20 epitope to which tosituzumab binds;
The antibody is ibritumomab, and the extracellular domain comprises a CD20 epitope to which the ibritumomab binds; or
5. The pharmaceutical composition according to configuration 4, wherein said antibody is ofatumumab and said extracellular domain comprises a CD20 epitope to which said ofatumumab binds.
(Configuration 7)
The T lymphocyte of any one of arrangements 1 to 3 further comprising a chimeric antigen receptor (CAR) that recognizes an antigen on a tumor cell.
(Configuration 8)
7. The T lymphocyte according to configuration 7, wherein said tumor cell is a solid tumor cell.
(Configuration 9)
The T lymphocyte according to configuration 7, wherein the tumor cell is a cell of hematologic cancer.
(Configuration 10)
Said antigens include Her2, prostate stem cell antigen, alpha-fetoprotein, carcinoembryonic antigen, cancer antigen-125, CA 19-9, calretinin, MUC-1, epithelial membrane protein, epithelial tumor antigen, tyrosinase, melanoma related antigen, CD34 , CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein, macroscopic cystic disease fluid protein, HMB-45 antigen, protein melan-A, myo-D1, muscle specific actin, neurofilament, 7. The T according to any one of arrangements 7 to 9, which is a neuron specific enolase, placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1 or pyruvate kinase isoenzyme M2 type in dimeric form. lymphocytes.
(Configuration 11)
7. The pharmaceutical composition according to any one of aspects 4 to 6, wherein said T lymphocytes further comprise a chimeric antigen receptor (CAR) that recognizes an antigen on tumor cells.
(Configuration 12)
Said antigens include Her2, prostate stem cell antigen, alpha-fetoprotein, carcinoembryonic antigen, cancer antigen-125, CA 19-9, calretinin, MUC-1, epithelial membrane protein, epithelial tumor antigen, tyrosinase, melanoma related antigen, CD34 , CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein, macroscopic cystic disease fluid protein, HMB-45 antigen, protein melan-A, myo-D1, muscle specific actin, neurofilament, 11. The pharmaceutical composition according to constitution 11, which is a neuron-specific enolase, placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1 or pyruvate kinase isozyme M type in dimeric form.
(Configuration 13)
5. The T lymphocyte of construction 3 wherein said antibody is rituximab and said extracellular domain comprises a CD20 epitope to which said rituximab binds.
(Configuration 14)
7. The pharmaceutical composition according to configuration 6, wherein said antibody is rituximab and said extracellular domain comprises a CD20 epitope to which said rituximab binds.

Claims (14)

T lymphocytes comprising an artificial cell death polypeptide comprising an apoptosis induction domain,
The artificial cell death polypeptide is a transmembrane protein comprising an extracellular domain comprising a CD20 epitope, a transmembrane domain, and an intracellular domain comprising the apoptosis induction domain, wherein the apoptosis induction domain is caspase 3, caspase 8 or caspase 9,
The polypeptide is dimerizable with an anti-CD20 antibody that binds to the CD20 epitope, and when the antibody dimerizes the polypeptide, the apoptosis induction signal is
The T lymphocytes, which are generated by T lymphocytes. 2. The antibody is approved by the U.S. Food and Drug Administration for any use.
Description T lymphocytes. Whether the antibody is rituximab and the extracellular domain comprises a CD20 epitope to which the rituximab binds;
Whether the antibody is tosituzumab and the extracellular domain comprises a CD20 epitope to which tosituzumab binds;
The antibody is ibritumomab, and the extracellular domain comprises a CD20 epitope to which the ibritumomab binds; or the antibody is ofatumumab and the extracellular domain is a CD20 epitope to which the OFATUMUM binds. The T lymphocyte according to claim 1 comprising. A pharmaceutical composition for killing T lymphocytes, wherein the T lymphocytes comprise a plurality of artificial cell death polypeptides each comprising an apoptosis induction domain, wherein the artificial cell death polypeptide comprises a CD20 epitope. A transmembrane protein comprising an exodomain, a transmembrane domain, and an intracellular domain comprising said apoptosis induction domain, said apoptosis induction domain is caspase 3, caspase 8 or caspase 9, said polypeptide is It is characterized that it can be dimerized using an anti-CD20 antibody that binds to a CD20 epitope, and that when the antibody dimerizes the polypeptide, an apoptosis induction signal is generated in the T lymphocytes. And wherein the plurality of artificial cell death polypeptides are dimerized and the collective apoptosis induction signal is sufficient to kill the T lymphocytes. Said pharmaceutical composition comprising an amount of said antibody sufficient to generate. 5. The antibody is approved by the U.S. Food and Drug Administration for any use.
Pharmaceutical composition as described. Whether the antibody is rituximab and the extracellular domain comprises a CD20 epitope to which the rituximab binds;
Whether the antibody is tosituzumab and the extracellular domain comprises a CD20 epitope to which tosituzumab binds;
The antibody is ibritumomab, and the extracellular domain comprises a CD20 epitope to which the ibritumomab binds; or the antibody is ofatumumab and the extracellular domain is a CD20 epitope to which the OFATUMUM binds. The pharmaceutical composition according to claim 4 comprising. The T lymphocyte according to any one of claims 1 to 3, further comprising a chimeric antigen receptor (CAR) that recognizes an antigen on tumor cells.   The T lymphocyte according to claim 7, wherein the tumor cell is a solid tumor cell.   The T lymphocyte according to claim 7, wherein the tumor cell is a blood cancer cell. Said antigens include Her2, prostate stem cell antigen, alpha-fetoprotein, oncofetal antigen, cancer antigen-
125, CA 19-9, calretinin, MUC-1, epithelial membrane protein, epithelial tumor antigen, tyrosinase,
Melanoma related antigen, CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein, macroscopic cystic disease protein, HMB-45 antigen, protein melan-A, myo-D1, muscle specific 10. The actin, neurofilament, neuron specific enolase, placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, or pyruvate kinase isozyme type M2 in dimeric form. Or a T lymphocyte as described in 1 above. The T lymphocytes further comprise a chimeric antigen receptor (CAR) that recognizes an antigen on tumor cells,
The pharmaceutical composition according to any one of claims 4 to 6. Said antigens include Her2, prostate stem cell antigen, alpha-fetoprotein, oncofetal antigen, cancer antigen-
125, CA 19-9, calretinin, MUC-1, epithelial membrane protein, epithelial tumor antigen, tyrosinase,
Melanoma related antigen, CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein, macroscopic cystic disease protein, HMB-45 antigen, protein melan-A, myo-D1, muscle specific The pharmaceutical composition according to claim 11, which is actin, neurofilament, neuron specific enolase, placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, or pyruvate kinase isoenzyme M2 type in dimeric form. object. The T lymphocyte of claim 3, wherein the antibody is rituximab and the extracellular domain comprises a CD20 epitope to which the rituximab binds. The pharmaceutical composition according to claim 6, wherein the antibody is rituximab, and the extracellular domain comprises a CD20 epitope to which the rituximab binds.